EP3184523B1 - Bromhydrate de n-((4,6-diméthyl-2-oxo-l,2-dihydropyridin-3-yl)méthyl)-5-(éthyl (tétrahydro-2h-pyran-4-yl)amino)-4-méthyl-4'-(morpholinométhyl)-[l,1'-biphényl]-3-carboxamide pour l'utilisation dans le traitement d'un trouble de prolifération cellulaire du système hématologique - Google Patents

Bromhydrate de n-((4,6-diméthyl-2-oxo-l,2-dihydropyridin-3-yl)méthyl)-5-(éthyl (tétrahydro-2h-pyran-4-yl)amino)-4-méthyl-4'-(morpholinométhyl)-[l,1'-biphényl]-3-carboxamide pour l'utilisation dans le traitement d'un trouble de prolifération cellulaire du système hématologique Download PDF

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EP3184523B1
EP3184523B1 EP16191716.6A EP16191716A EP3184523B1 EP 3184523 B1 EP3184523 B1 EP 3184523B1 EP 16191716 A EP16191716 A EP 16191716A EP 3184523 B1 EP3184523 B1 EP 3184523B1
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Prior art keywords
degrees
polymorph
cancer
compound
theta
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German (de)
English (en)
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EP3184523A1 (fr
Inventor
Kevin Wayne Kuntz
Kuan-Chun Huang
Hyeong Wook Choi
Kristen SANDERS
Steven Mathieu
Arani CHANDA
Frank Fang
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Eisai R&D Management Co Ltd
Epizyme Inc
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Eisai R&D Management Co Ltd
Epizyme Inc
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Priority to EP22200677.7A priority Critical patent/EP4190777A1/fr
Priority to EP19180479.8A priority patent/EP3628670B1/fr
Priority to SI201331566T priority patent/SI3184523T1/sl
Priority to DK19180479.8T priority patent/DK3628670T3/da
Priority to RSP20191175 priority patent/RS59392B1/sr
Application filed by Eisai R&D Management Co Ltd, Epizyme Inc filed Critical Eisai R&D Management Co Ltd
Priority to PL16191716T priority patent/PL3184523T3/pl
Publication of EP3184523A1 publication Critical patent/EP3184523A1/fr
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Publication of EP3184523B1 publication Critical patent/EP3184523B1/fr
Priority to HRP20191653TT priority patent/HRP20191653T1/hr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • a number of cancer deaths are caused by blood cancers including leukaemias, myelomas, and lymphomas.
  • lymphomas In 2012, almost 80,000 of cancer cases will be lymphomas, which will result in an estimated 20,000 deaths.
  • polymorph form of N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide hydrobromide
  • Polymorph A or "Polymorph A of N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide hydrobromide ").
  • the hydrobromide salt provided herein, as well as Polymorph A exhibit physical properties that can be exploited in order to obtain new pharmacological properties, and that
  • the hydrobromide is crystalline. In another embodiment, the hydrobromide is free of impurities. In another embodiment, the hydrobromide is a crystalline solid substantially free of amorphous N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide hydrobromide.
  • composition comprising the hydrobromide described above, and a pharmaceutically acceptable carrier or diluent.
  • the hydrobromide described above may be prepared using a method comprising combining N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide with hydrobromic acid.
  • Polymorph A of N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide can be defined according to its X-ray powder diffraction pattern. Accordingly, in one embodiment, the polymorph exhibits an X-ray powder diffraction pattern having one or more characteristic peaks expressed in degrees 2-theta at 3.9 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, and 22.0 +/- 0.3 degrees 2-theta.
  • the polymorph exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at 3.9 +/-0.3 degrees, 17.5 +/- 0.3 degrees, and 22.0 +/- 0.3 degrees 2-theta.
  • the polymorph exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at 3.9 +/- 0.3 degrees, 10.1 +/- 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20.6 +/- 0.3 degrees, 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0.3 degrees and 23.6 +/- 0.3 degrees 2-theta.
  • the polymorph exhibits an X-ray powder diffraction pattern substantially in accordance with Figure 1 .
  • the polymorph exhibits an X-ray powder diffraction pattern substantially in accordance with Table
  • Polymorph A can also be defined according to its differential scanning calorimetry thermogram.
  • the polymorph exhibits a differential scanning calorimetry thermogram having a characteristic peak expressed in units of °C at a temperature of 255 +/-5°C.
  • the polymorph exhibits a differential scanning calorimetry thermogram substantially in accordance with Figure 3 .
  • Polymorph A may be prepared using a method comprising combining N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide with hydrobromic acid.
  • a method of recrystallizing Polymorph A which comprises the following steps: (a) dissolving Polymorph A in a first solvent, and (b) adding a second solvent, such that said polymorph is recrystallized.
  • the first solvent is ethanol
  • the second solvent is MTBE.
  • the method comprises (a) dissolving Polymorph A in ethanol, (b) heating the mixture, (c) adding MTBE to the mixture, forming a precipitate comprising said polymorph, and filtering the precipitate such that said polymorph is recrystallized.
  • Described herein is a pharmaceutical composition
  • a pharmaceutical composition comprising Polymorph A, and a pharmaceutically acceptable carrier or diluent.
  • Also described herein is a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the hydrobromide compound described above, Polymorph A, or a pharmaceutical composition comprising either of these compounds.
  • a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the hydrobromide compound described above, Polymorph A, or a pharmaceutical composition comprising either of these compounds.
  • a variety of cancers can be treated, including non-Hodgkin's lymphoma or breast cancer.
  • Described herein is a method of inhibiting the histone methyltransferase activity of EZH2 in a subject in need thereof comprising administering to the subject an effective amount of the hydrobromide compound described above, Polymorph A, or a pharmaceutical composition comprising either of these compounds.
  • Described herein is a method of inhibiting the histone methyltransferase activity of EZH2 in vitro comprising administering the hydrobromide compound described above, or Polymorph A.
  • Described herein is the use of the hydrobromide compound described above, Polymorph A, or a pharmaceutical composition comprising either of these compounds, for the preparation of a medicament for the treatment of cancer in a subject in need thereof.
  • Compound I refers to N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide.
  • the hydrobromide of Compound I can be used to inhibit the histone methyltransferase activity of EZH2, either in a subject or in vitro.
  • the hydrobromide of Compound I can also be used to treat cancer in a subject in need thereof.
  • Compound I can be protonated at one or more of its basic sites, such as the morpholine, disubstituted aniline, and/or pyridone moieties. Accordingly, in certain embodiments, provided herein is the monohydrobromide, dihydrobromide, or trihydrobromide of Compound I. In one embodiment, provided herein is the monohydrobromide of Compound I. When the compound is the monohydrobromide, the compound may be protonated at any basic site. In a non-limiting embodiment, Compound I is protonated at the nitrogen of the morpholino substituent, providing a monohydrobromide of Compound I having the following structure:
  • This particular monohydrobromide can be referred to as "4-((3'-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-5'-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4'-methyl-[1,1'-biphenyl]-4-yl)methyl)morpholin-4-ium bromide.”
  • Figure 11 depicts the X-ray crystal structure of this particular salt form.
  • the hydrobromide of Compound I has a number of advantageous physical properties over its free base form, as well as other salts of the free base.
  • the hydrobromide of Compound I has low hygroscopicity compared to other salt forms of Compound I.
  • the compound For a compound to be effective in therapy, it is generally required that the compound be minimally hygroscopic. Drug forms that are highly hygroscopic may be unstable, as the drug form's dissolution rate may change as it is stored in settings with varying humidity. Also, hygroscopicity can impact large-scale handling and manufacturing of a compound, as it can be difficult to determine the true weight of a hygroscopic active agent when preparing a pharmaceutical composition comprising that agent.
  • the hydrobromide of Compound I has a low hygoscopicity compared to other salt forms of Compound I. As such, it can be stored over appreciable periods, and not suffer from detrimental changes in, for example, solubility, density, or even chemical composition.
  • the hydrobromide of Compound I can be produced in a highly crystalline form, which is useful in the preparation of pharmaceutical formulations, and will improve general handling, manipulation, and storage of the drug compound.
  • the crystalline form of the hydrobromide of Compound I is in a form referred to as "Polymorph A.”
  • polymorphism The ability of a substance to exist in more than one crystal form is defined as polymorphism; the different crystal forms of a particular substance are referred to as "polymorphs.”
  • polymorphism is affected by the ability of a molecule of a substance to change its conformation or to form different intermolecular or intra-molecular interactions, particularly hydrogen bonds, which is reflected in different atom arrangements in the crystal lattices of different polymorphs.
  • morphology which refers to the external shape of the crystal and the planes present, without reference to the internal structure. Crystals can display different morphology based on different conditions, such as, for example, growth rate, stirring, and the presence of impurities.
  • the different polymorphs of a substance may possess different energies of the crystal lattice and, thus, in solid state they can show different physical properties such as form, density, melting point, color, stability, solubility, dissolution rate, etc., which can, in turn, affect the stability, dissolution rate and/or bioavailability of a given polymorph and its suitability for use as a pharmaceutical and in pharmaceutical compositions.
  • Polymorph A is highly crystalline, and displays low hygroscopicity. Also, this polymorph can be obtained reproducibly, and slight changes in crystallization conditions do not result in different crystal forms.
  • Access to different polymorphs of the hydrobromide of Compound I is desirable for a number of reasons.
  • One such reason is that individual polymorphs can incorporate different impurities, or chemical residues, upon crystallization.
  • impurities can be removed during the process of converting Compound I into Polymorph A.
  • polymorph forms exhibiting compact crystal shapes possess advantages in terms of ease of filtration and ease of flow.
  • Polymorph A exhibits a compact crystal shape that therefore possesses these advantages.
  • Polymorph A is identifiable on the basis of characteristic peaks in an X-ray powder diffraction analysis.
  • X-ray powder diffraction also referred to as XRPD
  • Polymorph A exhibits an X-ray powder diffraction pattern having one or more characteristic peaks expressed in degrees 2-theta at 3.9 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, and 22.0 +/- 0.3 degrees 2-theta.
  • the polymorph exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at 3.9 +/- 0.3 degrees, 17.5 +/-0.3 degrees, and 22.0 +/- 0.3 degrees 2-theta.
  • Polymorph A exhibits an X-ray powder diffraction pattern having at least 5 characteristic peaks expressed in degrees 2-theta at 3.9 +/- 0.3 degrees, 10.1 +/- 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20.6 +/- 0.3 degrees, 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0.3 degrees and 23.6 +/-0.3 degrees 2-theta.
  • Polymorph A exhibits an X-ray powder diffraction pattern having at least 6 characteristic peaks expressed in degrees 2-theta at 3.9 +/- 0.3 degrees, 10.1 +/- 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20.6 +/- 0.3 degrees, 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0.3 degrees and 23.6 +/- 0.3 degrees 2-theta.
  • Polymorph A exhibits an X-ray powder diffraction pattern having at least 7 characteristic peaks expressed in degrees 2-theta at 3.9 +/-0.3 degrees, 10.1 +/- 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20.6 +/- 0.3 degrees, 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0.3 degrees and 23.6 +/- 0.3 degrees 2-theta.
  • Polymorph A exhibits an X-ray powder diffraction pattern having at least 8 characteristic peaks expressed in degrees 2-theta at 3.9 +/- 0.3 degrees, 10.1 +/- 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/-0.3 degrees, 20.6 +/- 0.3 degrees, 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0.3 degrees and 23.6 +/- 0.3 degrees 2-theta.
  • Polymorph A exhibits an X-ray powder diffraction pattern having at least 9 characteristic peaks expressed in degrees 2-theta at 3.9 +/- 0.3 degrees, 10.1 +/- 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20.6 +/- 0.3 degrees, 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0.3 degrees and 23.6 +/- 0.3 degrees 2-theta.
  • the polymorph exhibits an X-ray powder diffraction pattern having at least 10 characteristic peaks expressed in degrees 2-theta at 3.9 +/- 0.3 degrees, 10.1 +/- 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20.6 +/- 0.3 degrees, 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0.3 degrees and 23.6 +/- 0.3 degrees 2-theta.
  • the polymorph exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at 3.9 +/- 0.3 degrees, 14.3 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 23.3 +/- 0.3 degrees, and 23.6 +/- 0.3 degrees 2-theta.
  • the polymorph exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at 3.9 +/- 0.3 degrees, 10.1 +/- 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20.6 +/- 0.3 degrees, 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0.3 degrees and 23.6 +/-0.3 degrees 2-theta.
  • the polymorph exhibits an X-ray powder diffraction pattern substantially in accordance with Figure 1 .
  • the polymorph exhibits an X-ray powder diffraction pattern substantially in accordance with the 2-theta values listed in Table 1.
  • compositions comprising Polymorph A can be identified by comparison of the compositions' X-ray powder diffraction patterns to an X-ray powder diffraction pattern of Polymorph A. It will be appreciated that pharmaceutical compositions comprising Polymorph A may exhibit non-identical X-ray powder diffraction patterns as compared to an X-ray powder diffraction pattern of pure Polymorph A.
  • Polymorph A is identifiable on the basis of a characteristic peak observed in a differential scanning calorimetry thermogram.
  • Differential scanning calorimetry, or DSC is a thermoanalytical technique in which the difference in the amount of heat required to increase the temperature of a sample and reference is measured as a function of temperature.
  • Polymorph A exhibits a differential scanning calorimetry thermogram having a characteristic peak expressed in units of °C at a temperature of about 255 +/- 5°C.
  • Polymorph A exhibits a differential scanning calorimetry thermogram having a single endothermic peak observed at the temperature range of 250-255°C.
  • Polymorph A exhibits a differential scanning calorimetry thermogram substantially in accordance with Figure 3 .
  • Polymorph A may contain impurities.
  • impurities include undesired polymorph forms, or residual organic and inorganic molecules such as solvents, water or salts.
  • Polymorph A is free from impurities.
  • Polymorph A contains less than 10% by weight total impurities.
  • Polymorph A contains less than 5% by weight total impurities.
  • Polymorph A contains less than 1% by weight total impurities.
  • Polymorph A contains less than 0.1% by weight total impurities.
  • Polymorph A is a crystalline solid substantially free of amorphous Compound I hydrobromide.
  • substantially free of amorphous Compound I hydrobromide means that the compound contains no significant amount of amorphous Compound I hydrobromide.
  • at least about 95% by weight of crystalline Polymorph A is present.
  • at least about 99% by weight of crystalline Polymorph A is present.
  • Polymorph A is substantially free from Polymorph B.
  • the salt of the invention, and its crystal form Polymorph A can be found together with other substances or can be isolated.
  • the salt of the invention, or its crystal form is substantially isolated.
  • substantially isolated is meant that the salt or its crystal form is at least partially or substantially separated from the environment in which it was formed or detected.
  • Partial separation can include, for example, a composition enriched in the salt of the invention.
  • Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the hydrobromide of Compound I and Polymorph A. Methods for isolating compounds and their salts are routine in the art.
  • Both the hydrobromide of Compound I and Polymorph A can occur as any reasonable tautomer, or a mixture of reasonable tautomers.
  • tautomer refers to one of two or more structural isomers that exist in equilibrium and are readily converted from one isomeric form to another. Examples include keto-enol tautomers, such as acetone/propen-2-ol, and the like.
  • the hydrobromide of Compound I and Polymorph A can have one or more tautomers and therefore include various isomers, i.e., pyridin-2(1H)-one and the corresponding pyridin-2-ol. All such isomeric forms of these compounds are expressly included in the present invention.
  • the hydrobromide of Compound I, as well as Polymorph A can be prepared using known techniques.
  • a salt form is prepared by combining in solution the free base compound and an acid containing the anion of the salt form desired, and then isolating the solid salt product from the reaction solution (e.g., by crystallization, precipitation, evaporation, etc.).
  • Other salt-forming techniques can be employed.
  • step 3 This compound is then reacted under Suzuki coupling conditions in step 3 to form methyl 5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxylate (4), which is hydrolyzed to the corresponding acid (5) in step 4.
  • acid (5) is reacted under amide coupling reaction conditions with 3-(aminomethyl)-4,6-dimethyl-dihydro-pyridin-2(1H)-one hydrochloride to form Compound I.
  • Compound I can then be reacted with aqueous HBr to form the hydrobromide of Compound I.
  • the synthesis described above has a number of advantages. For example, it utilizes a number of intermediates that can be prepared in crystalline forms that can be isolated. By using crystalline intermediates, minimal purification techniques (e.g., chromatography) are necessary, leading to an overall improved yield of final Compound I.
  • minimal purification techniques e.g., chromatography
  • intermediate compound 1 in crystalline form.
  • intermediate compound 2 in crystalline form.
  • Figure 17 shows an X-ray powder diffraction pattern of crystalline compound 2.
  • the intermediate compound 5 is crystalline.
  • Figure 9 shows an X-ray powder diffraction pattern of crystalline compound 5.
  • compounds 2 and/or 5 are produced in substantially pure form without the use of chromatography. It will be appreciated by the skilled artisan that the crystallization of intermediates does not necessarily proceed effortlessly or efficiently.
  • Also provided herein is a method of preparing N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide comprising reacting 5-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxylic acid (5) with a salt of 3-(aminomethyl)-4,6-dimethyl-dihydro-pyridin-2(1H)-one.
  • (5) is in crystalline form.
  • Compound I can be reacted with aqueous HBr in the presence of an appropriate solvent to form Polymorph A, a particular crystal form of the hydrobromide.
  • Compound I is reacted with aqueous HBr in the presence of ethanol and ethyl acetate to form Polymorph A.
  • Polymorph A can be recrystallized by dissolving the polymorph in one or more solvents, optionally heating, followed by an optional cooling step, and then isolating the crystal structure, through, e.g., a filtering step.
  • an additional, different solvent can be added at any point in the process (before or after heating, before or after cooling, etc .) to produce the desired crystal structure.
  • a first solvent can be used to dissolve the polymorph compound, and then a second solvent (e.g ., an anti-solvent) can be added to cause the polymorph to precipitate from solution.
  • a second solvent e.g ., an anti-solvent
  • water is added to the first solvent to aid in dissolving the polymorph.
  • Non-limiting examples of solvents that can be used for the recrystallization of Polymorph A are as follows: methanol, ethanol, ethyl acetate, methyl tert-butyl ether, water, isopropyl alcohol, tetrahydrofuran, acetone, acetonitrile, and 2-methyltetrahydrofuran, as well as combinations thereof.
  • Non-limiting examples of solvent combinations that are useful for the recrystallization of Polymorph A are (solvent and anti-solvent, wherein water can be added to the first solvent to aid in dissolving the polymorph): methanol/water and ethyl acetate, isopropyl alcohol/water and ethyl acetate, tetrahydrofuran/water and ethyl acetate, acetone and ethyl acetate, acetonitrile/water and ethyl acetate, ethanol/water and methyl tert-butyl ether, isopropyl alcohol/water and methyl tert-butyl ether, ethanol/water and tetrahydrofuran, isopropyl alcohol/water and acetone, and ethanol/water and ethyl acetate.
  • the solvent combinations are methanol/water and ethyl acetate, isopropyl alcohol/water and ethyl acetate, ethanol/water and 2-methyltetrahydrofuran, and methanol/2-methyltetrahydrofuran.
  • Polymorph A is prepared using a method comprising combining N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide with hydrobromic acid.
  • a method of recrystallizing Polymorph A which comprises the following steps: (a) dissolving Polymorph A in a first solvent, and (b) adding a second solvent, such that said polymorph is recrystallized.
  • the first solvent is ethanol
  • the second solvent is MTBE.
  • the method comprises (a) dissolving Polymorph A in ethanol, (b) heating the mixture, (c) adding MTBE to the mixture, forming a precipitate comprising said polymorph, and filtering the precipitate such that said polymorph is recrystallized.
  • Described herein is a pharmaceutical composition comprising the hydrobromide of Compound I, and a pharmaceutically acceptable carrier or diluent. Also described herein is a pharmaceutical composition comprising Polymorph A, and a pharmaceutically acceptable carrier or diluent.
  • composition includes preparations suitable for administration to mammals, e.g., humans.
  • the compounds described herein are administered as pharmaceuticals to mammals, e.g., humans, they can be given per se or as a pharmaceutical composition containing, for example, 0.1% to 99.9% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • compositions described herein can be combined with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques.
  • pharmaceutically acceptable carrier may include any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980 ) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.
  • any conventional carrier medium is incompatible with the compounds such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition
  • materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatine; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; corn oil and soybean oil; glycols; such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide
  • the carrier may take a wide variety of forms depending on the form of the preparation desired for administration, e.g. oral, nasal, rectal, vaginal, parenteral (including intravenous injections or infusions).
  • oral, nasal, rectal, vaginal, parenteral including intravenous injections or infusions.
  • parenteral including intravenous injections or infusions.
  • any of the usual pharmaceutical media may be employed.
  • Usual pharmaceutical media include, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations (such as for example, suspensions, solutions, emulsions and elixirs); aerosols; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like, in the case of oral solid preparations (such as for example, powders, capsules, and tablets).
  • oral liquid preparations such as for example, suspensions, solutions, emulsions and elixirs
  • aerosols or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like
  • oral solid preparations such as for example, powders, capsules, and tablets.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, tocopherols, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, prop
  • compositions comprising the compounds may be formulated to have any concentration desired.
  • the composition is formulated such that it comprises at least a therapeutically effective amount.
  • the composition is formulated such that it comprises an amount that would not cause one or more unwanted side effects.
  • solid dosage forms are a preferred form for the pharmaceutical composition of the invention.
  • Solid dosage forms for oral administration such as capsules, tablets, pills, powders, and granules, are particularly preferred. If desired, tablets may be coated by techniques known to those in the art.
  • compositions include those suitable for oral, sublingual, nasal rectal, vaginal, topical, buccal and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route will depend on the nature and severity of the condition being treated.
  • the compositions may be conveniently presented in unit dosage form, and prepared by any of the methods well known in the art of pharmacy.
  • the pharmaceutical composition is formulated for oral administration in the form of a pill, capsule, lozenge or tablet.
  • the pharmaceutical composition is in the form of a suspension.
  • the compounds described herein are suitable as an active agent in pharmaceutical compositions that are efficacious particularly for treating EZH2-associated disorders, especially cancer.
  • the pharmaceutical composition in various embodiments has a pharmaceutically effective amount of the hydrobromide of Compound I or Polymorph A, along with other pharmaceutically acceptable excipients, carriers, fillers, diluents and the like.
  • a therapeutically or pharmaceutically "effective amount” is an amount of a compound (the hydrobromide of Compound I or Polymorph A), that when administered to a patient, ameliorates a symptom of a disease or condition, e.g., prevent the various morphological and somatic symptoms of cancer.
  • an effective amount of the hydrobromide of Compound I or Polymorph A is the amount sufficient to treat cancer in a subject. The amount can vary depending on such factors as the size and weight of the subject, the type of illness, or the particular compound of the invention.
  • the amount of the hydrobromide of Compound I or Polymorph A that constitutes an "effective amount” will vary depending on the compound, the disease state and its severity, the age of the patient to be treated, and the like. The effective amount can be determined routinely by one of ordinary skill in the art having regard to their knowledge and to this disclosure.
  • the regimen of administration can affect what constitutes a pharmaceutically effective amount.
  • the hydrobromide of Compound I or Polymorph A, and compositions comprising either of these compounds can be administered to the subject either prior to or after the onset of a disease. Further, several divided dosages, as well as staggered dosages can be administered daily or sequentially, or the dose can be continuously infused, or can be a bolus injection. Further, the dosages can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • the hydrobromide of Compound I, as well as Polymorph A inhibit the histone methyltransferase activity of EZH2 or a mutant thereof and, accordingly, in one aspect of the invention, these compounds disclosed herein are candidates for treating, or preventing certain conditions and diseases.
  • the present invention provides these compounds for treating conditions and diseases the course of which can be influenced by modulating the methylation status of histones or other proteins, wherein said methylation status is mediated at least in part by the activity of EZH2. Modulation of the methylation status of histones can in turn influence the level of expression of target genes activated by methylation, and/or target genes suppressed by methylation.
  • the use may include administering to a subject in need of such treatment, a therapeutically effective amount of a compound described herein.
  • the disorder in which EZH2-mediated protein methylation plays a part can be cancer or a precancerous condition.
  • the present invention further provides the hydrobromide of Compound I, as well as Polymorph A for use in the treatment of cancer or precancer the course of which can be influenced by modulating EZH2-mediated protein methylation, or, for the preparation of a medicament useful for the treatment of such cancer or pre-cancer.
  • Exemplary cancers that may be treated include lymphomas, including non-Hodgkin lymphoma, follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL); melanoma; and leukemia, including CML.
  • Exemplary precancerous condition includes myelodysplastic syndrome (MDS; formerly known as preleukemia).
  • these compounds for use in treating a lymphoma comprising administering to the subject in need thereof an effective amount of the hydrobromide of Compound I.
  • Polymorph A for treating a lymphoma.
  • the disorder can be cancer, e.g., cancer in which EZH2-mediated protein methylation plays a role.
  • compound (i.e., the hydrobromide of Compound I, as well as Polymorph A) for the preparation of a medicament useful for the prevention of a cell proliferative disorder associated, at least in part, with EZH2-mediated protein methylation.
  • the compounds described herein can be used to modulate protein (e.g., histone) methylation, e.g., to modulate histone methyltransferase or histone demethylase enzyme activity. At least some of the compounds can be used in vivo or in vitro for modulating protein methylation. Histone methylation has been reported to be involved in aberrant expression of certain genes in cancers, and in silencing of neuronal genes in non-neuronal cells. At least some compounds described herein are suitable candidates for treating these diseases, i.e., to decreases methylation or restores methylation to roughly its level in counterpart normal cells.
  • protein e.g., histone
  • Histone methylation has been reported to be involved in aberrant expression of certain genes in cancers, and in silencing of neuronal genes in non-neuronal cells.
  • At least some compounds described herein are suitable candidates for treating these diseases, i.e., to decreases methylation or restores methylation to roughly its level in
  • methylation modulators may be used for modulating cell proliferation. For example, in some cases excessive proliferation may be reduced with agents that decrease methylation, whereas insufficient proliferation may be stimulated with agents that increase methylation. Accordingly, diseases that may be treated by the compounds of the invention can include hyperproliferative diseases, such as benign cell growth and malignant cell growth.
  • a "subject in need thereof' is a subject having a disorder in which EZH2-mediated protein methylation plays a part, or a subject having an increased risk of developing such disorder relative to the population at large.
  • a subject in need thereof can have a precancerous condition.
  • a subject in need thereof has cancer.
  • a "subject” includes a mammal.
  • the mammal can be e.g., a human or appropriate non-human mammal, such as primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep or a pig.
  • the subject can also be a bird or fowl.
  • the mammal is a human.
  • cell proliferative disorder refers to conditions in which unregulated or abnormal growth, or both, of cells can lead to the development of an unwanted condition or disease, which may or may not be cancerous.
  • Exemplary cell proliferative disorders that may be treated with the compounds described herein encompass a variety of conditions wherein cell division is deregulated.
  • Exemplary cell proliferative disorder include, but are not limited to, neoplasms, benign tumors, malignant tumors, pre-cancerous conditions, in situ tumors, encapsulated tumors, metastatic tumors, liquid tumors, solid tumors, immunological tumors, hematological tumors, cancers, carcinomas, leukemias, lymphomas, sarcomas, and rapidly dividing cells.
  • a cell proliferative disorder includes a precancer or a precancerous condition.
  • a cell proliferative disorder includes cancer. The methods described herein may be used to treat or alleviate a symptom of cancer or to identify suitable candidates for such purposes.
  • cancer includes solid tumors, as well as, hematologic tumors and/or malignancies.
  • precancer cell or “precancerous cell” is a cell manifesting a cell proliferative disorder that is a precancer or a precancerous condition.
  • cancer cell or "cancerous cell” is a cell manifesting a cell proliferative disorder that is a cancer. Any reproducible means of measurement may be used to identify cancer cells or precancerous cells. Cancer cells or precancerous cells can be identified by histological typing or grading of a tissue sample ( e.g., a biopsy sample). Cancer cells or precancerous cells can be identified through the use of appropriate molecular markers.
  • non-cancerous conditions or disorders that may be treated using one or more compounds described herein include, but are not limited to, rheumatoid arthritis; inflammation; autoimmune disease; lymphoproliferative conditions; acromegaly; rheumatoid spondylitis; osteoarthritis; gout, other arthritic conditions; sepsis; septic shock; endotoxic shock; gram-negative sepsis; toxic shock syndrome; asthma; adult respiratory distress syndrome; chronic obstructive pulmonary disease; chronic pulmonary inflammation; inflammatory bowel disease; Crohn's disease; psoriasis; eczema; ulcerative colitis; pancreatic fibrosis; hepatic fibrosis; acute and chronic renal disease; irritable bowel syndrome; pyresis; restenosis; cerebral malaria; stroke and ischemic injury; neural trauma; Alzheimer's disease; Huntington's disease; Parkinson's disease; acute and chronic pain; allergic rhinitis; allergic
  • Exemplary cancers that can be treated using one or more compounds described herein include, but are not limited to, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, anorectal cancer, cancer of the anal canal, appendix cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, skin cancer (non-melanoma), biliary cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, urinary bladder cancer, bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodeimal tumors, visual pathway and hypothalamic glioma, breast
  • a "cell proliferative disorder of the hematologic system” is a cell proliferative disorder involving cells of the hematologic system.
  • a cell proliferative disorder of the hematologic system can include lymphoma, leukemia, myeloid neoplasms, mast cell neoplasms, myelodysplasia, benign monoclonal gammopathy, lymphomatoid granulomatosis, lymphomatoid papulosis, polycythemia vera, chronic myelocytic leukemia, agnogenic myeloid metaplasia, and essential thrombocythemia.
  • a cell proliferative disorder of the hematologic system can include hyperplasia, dysplasia, and metaplasia of cells of the hematologic system.
  • compositions described herein may be used to treat a cancer selected from the group consisting of a hematologic cancer or a hematologic cell proliferative disorder, or used to identify suitable candidates for such purposes.
  • a hematologic cancer can include multiple myeloma, lymphoma (including Hodgkin's lymphoma, non-Hodgkin's lymphoma, childhood lymphomas, and lymphomas of lymphocytic and cutaneous origin), leukemia (including childhood leukemia, hairy-cell leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia, and mast cell leukemia), myeloid neoplasms and mast cell neoplasms.
  • lymphoma including Hodgkin's lymphoma, non-Hodgkin's lymphoma, childhood lymphomas, and lymphomas of lymphocytic and cutaneous origin
  • leukemia including childhood leukemia, hairy-cell leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, chronic lymphocy
  • a "cell proliferative disorder of the lung” is a cell proliferative disorder involving cells of the lung.
  • Cell proliferative disorders of the lung can include all forms of cell proliferative disorders affecting lung cells.
  • Cell proliferative disorders of the lung can include lung cancer, a precancer or precancerous condition of the lung, benign growths or lesions of the lung, and malignant growths or lesions of the lung, and metastatic lesions in tissue and organs in the body other than the lung.
  • compositions described herein may be used to treat lung cancer or cell proliferative disorders of the lung, or used to identify suitable candidates for such purposes.
  • Lung cancer can include all forms of cancer of the lung.
  • Lung cancer can include malignant lung neoplasms, carcinoma in situ, typical carcinoid tumors, and atypical carcinoid tumors.
  • Lung cancer can include small cell lung cancer ("SCLC"), non-small cell lung cancer ("NSCLC”), squamous cell carcinoma, adenocarcinoma, small cell carcinoma, large cell carcinoma, adenosquamous cell carcinoma, and mesothelioma.
  • Lung cancer can include "scar carcinoma,” bronchioalveolar carcinoma, giant cell carcinoma, spindle cell carcinoma, and large cell neuroendocrine carcinoma.
  • Lung cancer can include lung neoplasms having histologic and ultrastructual heterogeneity (e.g., mixed cell types).
  • Cell proliferative disorders of the lung can include all forms of cell proliferative disorders affecting lung cells.
  • Cell proliferative disorders of the lung can include lung cancer, precancerous conditions of the lung.
  • Cell proliferative disorders of the lung can include hyperplasia, metaplasia, and dysplasia of the lung.
  • Cell proliferative disorders of the lung can include asbestos-induced hyperplasia, squamous metaplasia, and benign reactive mesothelial metaplasia.
  • Cell proliferative disorders of the lung can include replacement of columnar epithelium with stratified squamous epithelium, and mucosal dysplasia.
  • Prior lung diseases that may predispose individuals to development of cell proliferative disorders of the lung can include chronic interstitial lung disease, necrotizing pulmonary disease, scleroderma, rheumatoid disease, sarcoidosis, interstitial pneumonitis, tuberculosis, repeated pneumonias, idiopathic pulmonary fibrosis, granulomata, asbestosis, fibrosing alveolitis, and Hodgkin's disease.
  • a "cell proliferative disorder of the colon” is a cell proliferative disorder involving cells of the colon.
  • the cell proliferative disorder of the colon is colon cancer.
  • compositions described herein may be used to treat colon cancer or cell proliferative disorders of the colon, or used to identify suitable candidates for such purposes.
  • Colon cancer can include all forms of cancer of the colon.
  • Colon cancer can include sporadic and hereditary colon cancers.
  • Colon cancer can include malignant colon neoplasms, carcinoma in situ, typical carcinoid tumors, and atypical carcinoid tumors.
  • Colon cancer can include adenocarcinoma, squamous cell carcinoma, and adenosquamous cell carcinoma.
  • Colon cancer can be associated with a hereditary syndrome selected from the group consisting of hereditary nonpolyposis colorectal cancer, familial adenomatous polyposis, Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndrome and juvenile polyposis.
  • Colon cancer can be caused by a hereditary syndrome selected from the group consisting of hereditary nonpolyposis colorectal cancer, familial adenomatous polyposis, Gardner's syndrome, Koz-Jeghers syndrome, Turcot's syndrome and juvenile polyposis.
  • Cell proliferative disorders of the colon can include all forms of cell proliferative disorders affecting colon cells.
  • Cell proliferative disorders of the colon can include colon cancer, precancerous conditions of the colon, adenomatous polyps of the colon and metachronous lesions of the colon.
  • a cell proliferative disorder of the colon can include adenoma.
  • Cell proliferative disorders of the colon can be characterized by hyperplasia, metaplasia, and dysplasia of the colon.
  • Prior colon diseases that may predispose individuals to development of cell proliferative disorders of the colon can include prior colon cancer.
  • Current disease that may predispose individuals to development of cell proliferative disorders of the colon can include Crohn's disease and ulcerative colitis.
  • a cell proliferative disorder of the colon can be associated with a mutation in a gene selected from the group consisting of p53, ras, FAP and DCC.
  • An individual can have an elevated risk of developing a cell proliferative disorder of the colon due to the presence of a mutation in a gene selected from the group consisting of p53, ras, FAP and DCC.
  • a "cell proliferative disorder of the pancreas” is a cell proliferative disorder involving cells of the pancreas.
  • Cell proliferative disorders of the pancreas can include all forms of cell proliferative disorders affecting pancreatic cells.
  • Cell proliferative disorders of the pancreas can include pancreas cancer, a precancer or precancerous condition of the pancreas, hyperplasia of the pancreas, and dysaplasia of the pancreas, benign growths or lesions of the pancreas, and malignant growths or lesions of the pancreas, and metastatic lesions in tissue and organs in the body other than the pancreas.
  • Pancreatic cancer includes all forms of cancer of the pancreas.
  • Pancreatic cancer can include ductal adenocarcinoma, adenosquamous carcinoma, pleomorphic giant cell carcinoma, mucinous adenocarcinoma, osteoclast-like giant cell carcinoma, mucinous cystadenocarcinoma, acinar carcinoma, unclassified large cell carcinoma, small cell carcinoma, pancreatoblastoma, papillary neoplasm, mucinous cystadenoma, papillary cystic neoplasm, and serous cystadenoma.
  • Pancreatic cancer can also include pancreatic neoplasms having histologic and ultrastructual heterogeneity (e.g ., mixed cell types).
  • a "cell proliferative disorder of the prostate” is a cell proliferative disorder involving cells of the prostate.
  • Cell proliferative disorders of the prostate can include all forms of cell proliferative disorders affecting prostate cells.
  • Cell proliferative disorders of the prostate can include prostate cancer, a precancer or precancerous condition of the prostate, benign growths or lesions of the prostate, and malignant growths or lesions of the prostate, and metastatic lesions in tissue and organs in the body other than the prostate.
  • Cell proliferative disorders of the prostate can include hyperplasia, metaplasia, and dysplasia of the prostate.
  • a "cell proliferative disorder of the skin” is a cell proliferative disorder involving cells of the skin.
  • Cell proliferative disorders of the skin can include all forms of cell proliferative disorders affecting skin cells.
  • Cell proliferative disorders of the skin can include a precancer or precancerous condition of the skin, benign growths or lesions of the skin, melanoma, malignant melanoma and other malignant growths or lesions of the skin, and metastatic lesions in tissue and organs in the body other than the skin.
  • Cell proliferative disorders of the skin can include hyperplasia, metaplasia, and dysplasia of the skin.
  • a "cell proliferative disorder of the ovary” is a cell proliferative disorder involving cells of the ovary.
  • Cell proliferative disorders of the ovary can include all forms of cell proliferative disorders affecting cells of the ovary.
  • Cell proliferative disorders of the ovary can include a precancer or precancerous condition of the ovary, benign growths or lesions of the ovary, ovarian cancer, malignant growths or lesions of the ovary, and metastatic lesions in tissue and organs in the body other than the ovary.
  • Cell proliferative disorders of the skin can include hyperplasia, metaplasia, and dysplasia of cells of the ovary.
  • a "cell proliferative disorder of the breast” is a cell proliferative disorder involving cells of the breast.
  • Cell proliferative disorders of the breast can include all forms of cell proliferative disorders affecting breast cells.
  • Cell proliferative disorders of the breast can include breast cancer, a precancer or precancerous condition of the breast, benign growths or lesions of the breast, and malignant growths or lesions of the breast, and metastatic lesions in tissue and organs in the body other than the breast.
  • Cell proliferative disorders of the breast can include hyperplasia, metaplasia, and dysplasia of the breast.
  • a cell proliferative disorder of the breast can be a precancerous condition of the breast.
  • Compositions described herein may be used to treat a precancerous condition of the breast.
  • a precancerous condition of the breast can include atypical hyperplasia of the breast, ductal carcinoma in situ (DCIS), intraductal carcinoma, lobular carcinoma in situ (LCIS), lobular neoplasia, and stage 0 or grade 0 growth or lesion of the breast ( e.g. , stage 0 or grade 0 breast cancer, or carcinoma in situ ).
  • a precancerous condition of the breast can be staged according to the TNM classification scheme as accepted by the American Joint Committee on Cancer (AJCC), where the primary tumor (T) has been assigned a stage of T0 or Tis; and where the regional lymph nodes (N) have been assigned a stage of N0; and where distant metastasis (M) has been assigned a stage of M0.
  • AJCC American Joint Committee on Cancer
  • the cell proliferative disorder of the breast can be breast cancer.
  • compositions described herein may be used to treat breast cancer, or used to identify suitable candidates for such purposes.
  • Breast cancer may include all forms of cancer of the breast.
  • Breast cancer can include primary epithelial breast cancers.
  • Breast cancer can include cancers in which the breast is involved by other tumors such as lymphoma, sarcoma or melanoma.
  • Breast cancer can include carcinoma of the breast, ductal carcinoma of the breast, lobular carcinoma of the breast, undifferentiated carcinoma of the breast, cystosarcoma phyllodes of the breast, angiosarcoma of the breast, and primary lymphoma of the breast.
  • Breast cancer can include Stage I, II, IIIA, IIIB, IIIC and IV breast cancer.
  • Ductal carcinoma of the breast can include invasive carcinoma, invasive carcinoma in situ with predominant intraductal component, inflammatory breast cancer, and a ductal carcinoma of the breast with a histologic type selected from the group consisting of comedo, mucinous (colloid), medullary, medullary with lymphcytic infiltrate, papillary, scirrhous, and tubular.
  • Lobular carcinoma of the breast can include invasive lobular carcinoma with predominant in situ component, invasive lobular carcinoma, and infiltrating lobular carcinoma.
  • Breast cancer can include Paget's disease, Paget's disease with intraductal carcinoma, and Paget's disease with invasive ductal carcinoma.
  • Breast cancer can include breast neoplasms having histologic and ultrastructual heterogeneity (e.g ., mixed cell types).
  • a breast cancer that is to be treated can include familial breast cancer.
  • a breast cancer that is to be treated can include sporadic breast cancer.
  • a breast cancer that is to be treated can arise in a male subject.
  • a breast cancer that is to be treated can arise in a female subject.
  • a breast cancer that is to be treated can arise in a premenopausal female subject or a postmenopausal female subject.
  • a breast cancer that is to be treated can arise in a subject equal to or older than 30 years old, or a subject younger than 30 years old.
  • a breast cancer that is to be treated has arisen in a subject equal to or older than 50 years old, or a subject younger than 50 years old.
  • a breast cancer that is to be treated can arise in a subject equal to or older than 70 years old, or a subject younger than 70 years old.
  • a breast cancer that is to be treated can be typed to identify a familial or spontaneous mutation in BRCA1, BRCA2, or p53.
  • a breast cancer that is to be treated can be typed as having a HER2/neu gene amplification, as overexpressing HER2/neu, or as having a low, intermediate or high level of HER2/neu expression.
  • a breast cancer that is to be treated can be typed for a marker selected from the group consisting of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2, Ki-67, CA15-3, CA 27-29, and c-Met.
  • ER estrogen receptor
  • PR progesterone receptor
  • Ki-67 human epidermal growth factor receptor-2
  • Ki-67 Ki-67
  • CA15-3 CA 27-29
  • CA 27-29 CA 27-29
  • c-Met c-Met
  • a breast cancer that is to be treated can be typed as ER-negative or ER-positive.
  • ER-typing of a breast cancer may be performed by any reproducible means. ER-typing of a breast cancer may be performed as set forth in Onkologie 27: 175-179 (2004 ).
  • a breast cancer that is to be treated can be typed as PR-unknown, PR-rich, or PR-poor.
  • a breast cancer that is to be treated can be typed as PR-negative or PR-positive.
  • a breast cancer that is to be treated can be typed as receptor positive or receptor negative.
  • a breast cancer that is to be treated can be typed as being associated with elevated blood levels of CA 15-3, or CA 27-29, or both.
  • a breast cancer that is to be treated can include a localized tumor of the breast.
  • a breast cancer that is to be treated can include a tumor of the breast that is associated with a negative sentinel lymph node (SLN) biopsy.
  • a breast cancer that is to be treated can include a tumor of the breast that is associated with a positive sentinel lymph node (SLN) biopsy.
  • a breast cancer that is to be treated can include a tumor of the breast that is associated with one or more positive axillary lymph nodes, where the axillary lymph nodes have been staged by any applicable method.
  • a breast cancer that is to be treated can include a tumor of the breast that has been typed as having nodal negative status (e.g., node-negative) or nodal positive status (e.g., node-positive).
  • a breast cancer that is to be treated can include a tumor of the breast that has metastasized to other locations in the body.
  • a breast cancer that is to be treated can be classified as having metastasized to a location selected from the group consisting of bone, lung, liver, or brain.
  • a breast cancer that is to be treated can be classified according to a characteristic selected from the group consisting of metastatic, localized, regional, local-regional, locally advanced, distant, multicentric, bilateral, ipsilateral, contralateral, newly diagnosed, recurrent, and inoperable.
  • a compound described herein may be used to treat or prevent a cell proliferative disorder of the breast, or to treat or prevent breast cancer, in a subject having an increased risk of developing breast cancer relative to the population at large, or used to identify suitable candidates for such purposes.
  • a subject with an increased risk of developing breast cancer relative to the population at large is a female subject with a family history or personal history of breast cancer.
  • a subject with an increased risk of developing breast cancer relative to the population at large is a female subject having a germ-line or spontaneous mutation in BRCA1 or BRCA2, or both.
  • a subject with an increased risk of developing breast cancer relative to the population at large is a female subject with a family history of breast cancer and a germ-line or spontaneous mutation in BRCA1 or BRCA2, or both.
  • a subject with an increased risk of developing breast cancer relative to the population at large is a female who is greater than 30 years old, greater than 40 years old, greater than 50 years old, greater than 60 years old, greater than 70 years old, greater than 80 years old, or greater than 90 years old.
  • a subject with an increased risk of developing breast cancer relative to the population at large is a subject with atypical hyperplasia of the breast, ductal carcinoma in situ (DCIS), intraductal carcinoma, lobular carcinoma in situ (LCIS), lobular neoplasia, or a stage 0 growth or lesion of the breast ( e.g., stage 0 or grade 0 breast cancer, or carcinoma in situ ).
  • a breast cancer that is to be treated can be histologically graded according to the Scarff-Bloom-Richardson system, wherein a breast tumor has been assigned a mitosis count score of 1, 2, or 3; a nuclear pleiomorphism score of 1, 2, or 3; a tubule formation score of 1, 2, or 3; and a total Scarff-Bloom-Richardson score of between 3 and 9.
  • a breast cancer that is to be treated can be assigned a tumor grade according to the International Consensus Panel on the Treatment of Breast Cancer selected from the group consisting of grade 1, grade 1-2, grade 2, grade 2-3, or grade 3.
  • Described herein is a method of treating breast cancer comprising administering to a subject in need thereof an effective amount of the hydrobromide of Compound I.
  • Also described herein is a method of treating breast cancer comprising administering to a subject in need thereof an effective amount of Polymorph A.
  • a cancer that is to be treated can be staged according to the American Joint Committee on Cancer (AJCC) TNM classification system, where the tumor (T) has been assigned a stage of TX, T1, T1mic, T1a, T1b, T1c, T2, T3, T4, T4a, T4b, T4c, or T4d; and where the regional lymph nodes (N) have been assigned a stage of NX, N0, N1, N2, N2a, N2b, N3, N3a, N3b, or N3c; and where distant metastasis (M) can be assigned a stage of MX, M0, or M1.
  • AJCC American Joint Committee on Cancer
  • a cancer that is to be treated can be staged according to an American Joint Committee on Cancer (AJCC) classification as Stage I, Stage IIA, Stage IIB, Stage IIIA, Stage IIIB, Stage IIIC, or Stage IV.
  • AJCC American Joint Committee on Cancer
  • a cancer that is to be treated can be assigned a grade according to an AJCC classification as Grade GX (e.g., grade cannot be assessed), Grade 1, Grade 2, Grade 3 or Grade 4.
  • a cancer that is to be treated can be staged according to an AJCC pathologic classification (pN) of pNX, pN0, PN0 (I-), PN0 (I+), PN0 (mol-), PN0 (mol+), PN1, PN1(mi), PN1a, PN1b, PN1c, pN2, pN2a, pN2b, pN3, pN3a, pN3b, or pN3c.
  • pN AJCC pathologic classification
  • a cancer that is to be treated can include a tumor that has been determined to be less than or equal to about 2 centimeters in diameter.
  • a cancer that is to be treated can include a tumor that has been determined to be from about 2 to about 5 centimeters in diameter.
  • a cancer that is to be treated can include a tumor that has been determined to be greater than or equal to about 3 centimeters in diameter.
  • a cancer that is to be treated can include a tumor that has been determined to be greater than 5 centimeters in diameter.
  • a cancer that is to be treated can be classified by microscopic appearance as well differentiated, moderately differentiated, poorly differentiated, or undifferentiated.
  • a cancer that is to be treated can be classified by microscopic appearance with respect to mitosis count (e.g., amount of cell division) or nuclear pleiomorphism ( e.g., change in cells).
  • a cancer that is to be treated can be classified by microscopic appearance as being associated with areas of necrosis ( e.g ., areas of dying or degenerating cells).
  • a cancer that is to be treated can be classified as having an abnormal karyotype, having an abnormal number of chromosomes, or having one or more chromosomes that are abnormal in appearance.
  • a cancer that is to be treated can be classified as being aneuploid, triploid, tetraploid, or as having an altered ploidy.
  • a cancer that is to be treated can be classified as having a chromosomal translocation, or a deletion or duplication of an entire chromosome, or a region of deletion, duplication or amplification of a portion of a chromosome.
  • a cancer that is to be treated can be evaluated by DNA cytometry, flow cytometry, or image cytometry.
  • a cancer that is to be treated can be typed as having 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of cells in the synthesis stage of cell division ( e.g ., in S phase of cell division).
  • a cancer that is to be treated can be typed as having a low S-phase fraction or a high S-phase fraction.
  • a "normal cell” is a cell that cannot be classified as part of a "cell proliferative disorder”.
  • a normal cell lacks unregulated or abnormal growth, or both, that can lead to the development of an unwanted condition or disease.
  • a normal cell possesses normally functioning cell cycle checkpoint control mechanisms.
  • contacting a cell refers to a condition in which a compound or other composition of matter is in direct contact with a cell, or is close enough to induce a desired biological effect in a cell.
  • candidate compound refers to a compound described herein (i.e., the hydrobromide of Compound I, as well as Polymorph A) that has been or will be tested in one or more in vitro or in vivo biological assays, in order to determine if that compound is likely to elicit a desired biological or medical response in a cell, tissue, system, animal or human that is being sought by a researcher or clinician.
  • a candidate compound is a compound described herein.
  • the biological or medical response can be the treatment of cancer.
  • the biological or medical response can be treatment or prevention of a cell proliferative disorder.
  • the biological response or effect can also include a change in cell proliferation or growth that occurs in vitro or in an animal model, as well as other biological changes that are observable in vitro.
  • In vitro or in vivo biological assays can include, but are not limited to, enzymatic activity assays, electrophoretic mobility shift assays, reporter gene assays, in vitro cell viability assays, and the assays described herein.
  • monotherapy refers to the administration of a single active or therapeutic compound to a subject in need thereof.
  • monotherapy will involve administration of a therapeutically effective amount of an active compound.
  • cancer monotherapy with one of the compound described herein (i.e., the hydrobromide of Compound I, as well as Polymorph A) to a subject in need of treatment of cancer.
  • Monotherapy may be contrasted with combination therapy, in which a combination of multiple active compounds is administered, preferably with each component of the combination present in a therapeutically effective amount.
  • monotherapy with a compound of described herein is more effective than combination therapy in inducing a desired biological effect.
  • treating describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a compound described herein (i.e., the hydrobromide of Compound I, as well as Polymorph A) to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder.
  • a compound described herein i.e., the hydrobromide of Compound I, as well as Polymorph A
  • the term “treat” can also include treatment of a cell in vitro or an animal model.
  • a compound described herein i.e., the hydrobromide of Compound I, as well as Polymorph A
  • "preventing” or “prevent” describes reducing or eliminating the onset of the symptoms or complications of the disease, condition or disorder.
  • the term "alleviate” is meant to describe a process by which the severity of a sign or symptom of a disorder is decreased.
  • a sign or symptom can be alleviated without being eliminated.
  • the administration of pharmaceutical compositions described herein may lead to the elimination of a sign or symptom, however, elimination is not required.
  • Effective dosages are expected to decrease the severity of a sign or symptom.
  • a sign or symptom of a disorder such as cancer, which can occur in multiple locations, is alleviated if the severity of the cancer is decreased within at least one of multiple locations.
  • severity is meant to describe the potential of cancer to transform from a precancerous, or benign, state into a malignant state.
  • severity is meant to describe a cancer stage, for example, according to the TNM system (accepted by the International Union against Cancer (UICC) and the American Joint Committee on Cancer (AJCC)) or by other art-recognized methods.
  • TNM system accepted by the International Union against Cancer (UICC) and the American Joint Committee on Cancer (AJCC)
  • UNM system International Union against Cancer
  • AJCC American Joint Committee on Cancer
  • Cancer stage refers to the extent or severity of the cancer, based on factors such as the location of the primary tumor, tumor size, number of tumors, and lymph node involvement (spread of cancer into lymph nodes).
  • Tumor grade is a system used to classify cancer cells in terms of how abnormal they look under a microscope and how quickly the tumor is likely to grow and spread. Many factors are considered when determining tumor grade, including the structure and growth pattern of the cells. The specific factors used to determine tumor grade vary with each type of cancer. Severity also describes a histologic grade, also called differentiation, which refers to how much the tumor cells resemble normal cells of the same tissue type (see, National Cancer Institute, www.cancer.gov). Furthermore, severity describes a nuclear grade, which refers to the size and shape of the nucleus in tumor cells and the percentage of tumor cells that are dividing (see, National Cancer Institute, www.cancer.gov).
  • Severity describes the degree to which a tumor has secreted growth factors, degraded the extracellular matrix, become vascularized, lost adhesion to juxtaposed tissues, or metastasized. Moreover, severity describes the number of locations to which a primary tumor has metastasized. Finally, severity includes the difficulty of treating tumors of varying types and locations. For example, inoperable tumors, those cancers which have greater access to multiple body systems (hematological and immunological tumors), and those which are the most resistant to traditional treatments are considered most severe.
  • symptom is defined as an indication of disease, illness, injury, or that something is not right in the body. Symptoms are felt or noticed by the individual experiencing the symptom, but may not easily be noticed by others. Others are defined as non-health-care professionals.
  • signs are also defined as an indication that something is not right in the body. But signs are defined as things that can be seen by a doctor, nurse, or other health care professional.
  • Cancer is a group of diseases that may cause almost any sign or symptom. The signs and symptoms will depend on where the cancer is, the size of the cancer, and how much it affects the nearby organs or structures. If a cancer spreads (metastasizes), then symptoms may appear in different parts of the body.
  • cancer As a cancer grows, it begins to push on nearby organs, blood vessels, and nerves. This pressure creates some of the signs and symptoms of cancer. If the cancer is in a critical area, such as certain parts of the brain, even the smallest tumor can cause early symptoms.
  • pancreas cancers for example, do not usually grow large enough to be felt from the outside of the body. Some pancreatic cancers do not cause symptoms until they begin to grow around nearby nerves (this causes a backache). Others grow around the bile duct, which blocks the flow of bile and leads to a yellowing of the skin known as jaundice. By the time a pancreatic cancer causes these signs or symptoms, it has usually reached an advanced stage.
  • a cancer may also cause symptoms such as fever, fatigue, or weight loss. This may be because cancer cells use up much of the body's energy supply or release substances that change the body's metabolism. Or the cancer may cause the immune system to react in ways that produce these symptoms.
  • cancer cells release substances into the bloodstream that cause symptoms not usually thought to result from cancers.
  • some cancers of the pancreas can release substances which cause blood clots to develop in veins of the legs.
  • Some lung cancers make hormone-like substances that affect blood calcium levels, affecting nerves and muscles and causing weakness and dizziness
  • Cancer presents several general signs or symptoms that occur when a variety of subtypes of cancer cells are present. Most people with cancer will lose weight at some time with their disease. An unexplained (unintentional) weight loss of 10 pounds or more may be the first sign of cancer, particularly cancers of the pancreas, stomach, esophagus, or lung.
  • Fever is very common with cancer, but is more often seen in advanced disease. Almost all patients with cancer will have fever at some time, especially if the cancer or its treatment affects the immune system and makes it harder for the body to fight infection. Less often, fever may be an early sign of cancer, such as with leukemia or lymphoma.
  • Fatigue may be an important symptom as cancer progresses. It may happen early, though, in cancers such as with leukemia, or if the cancer is causing an ongoing loss of blood, as in some colon or stomach cancers.
  • cancer subtypes present specific signs or symptoms. Changes in bowel habits or bladder function could indicate cancer. Long-term constipation, diarrhea, or a change in the size of the stool may be a sign of colon cancer. Pain with urination, blood in the urine, or a change in bladder function (such as more frequent or less frequent urination) could be related to bladder or prostate cancer.
  • Skin cancers may bleed and look like sores that do not heal.
  • a long-lasting sore in the mouth could be an oral cancer, especially in patients who smoke, chew tobacco, or frequently drink alcohol. Sores on the penis or vagina may either be signs of infection or an early cancer.
  • Unusual bleeding or discharge could indicate cancer. Unusual bleeding can happen in either early or advanced cancer. Blood in the sputum (phlegm) may be a sign of lung cancer. Blood in the stool (or a dark or black stool) could be a sign of colon or rectal cancer. Cancer of the cervix or the endometrium (lining of the uterus) can cause vaginal bleeding. Blood in the urine may be a sign of bladder or kidney cancer. A bloody discharge from the nipple may be a sign of breast cancer.
  • a thickening or lump in the breast or in other parts of the body could indicate the presence of a cancer. Many cancers can be felt through the skin, mostly in the breast, testicle, lymph nodes (glands), and the soft tissues of the body. A lump or thickening may be an early or late sign of cancer. Any lump or thickening could be indicative of cancer, especially if the formation is new or has grown in size.
  • Indigestion or trouble swallowing could indicate cancer. While these symptoms commonly have other causes, indigestion or swallowing problems may be a sign of cancer of the esophagus, stomach, or pharynx (throat).
  • Wart or mole could be indicative of cancer. Any wart, mole, or freckle that changes in color, size, or shape, or loses its definite borders indicates the potential development of cancer.
  • the skin lesion may be a melanoma.
  • a persistent cough or hoarseness could be indicative of cancer.
  • a cough that does not go away may be a sign of lung cancer.
  • Hoarseness can be a sign of cancer of the larynx (voice box) or thyroid.
  • Treating cancer can result in a reduction in size of a tumor.
  • a reduction in size of a tumor may also be referred to as "tumor regression".
  • tumor size is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor size is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75% or greater.
  • Size of a tumor may be measured by any reproducible means of measurement. The size of a tumor may be measured as a diameter of the tumor.
  • Treating cancer can result in a reduction in tumor volume.
  • tumor volume is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor volume is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75% or greater.
  • Tumor volume may be measured by any reproducible means of measurement.
  • Treating cancer results in a decrease in number of tumors.
  • tumor number is reduced by 5% or greater relative to number prior to treatment; more preferably, tumor number is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%.
  • Number of tumors may be measured by any reproducible means of measurement.
  • the number of tumors may be measured by counting tumors visible to the naked eye or at a specified magnification.
  • the specified magnification is 2x, 3x, 4x, 5x, 10x, or 50x.
  • Treating cancer can result in a decrease in number of metastatic lesions in other tissues or organs distant from the primary tumor site.
  • the number of metastatic lesions is reduced by 5% or greater relative to number prior to treatment; more preferably, the number of metastatic lesions is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%.
  • the number of metastatic lesions may be measured by any reproducible means of measurement.
  • the number of metastatic lesions may be measured by counting metastatic lesions visible to the naked eye or at a specified magnification.
  • the specified magnification is 2x, 3x, 4x, 5x, 10x, or 50x.
  • Treating cancer can result in an increase in average survival time of a population of treated subjects in comparison to a population receiving carrier alone.
  • the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days.
  • An increase in average survival time of a population may be measured by any reproducible means.
  • An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound.
  • An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.
  • Treating cancer can result in an increase in average survival time of a population of treated subjects in comparison to a population of untreated subjects.
  • the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days.
  • An increase in average survival time of a population may be measured by any reproducible means.
  • An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound.
  • An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.
  • Treating cancer can result in increase in average survival time of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound described herein.
  • the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days.
  • An increase in average survival time of a population may be measured by any reproducible means.
  • An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound.
  • An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.
  • Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving carrier alone. Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to an untreated population. Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound described herein.
  • the mortality rate is decreased by more than 2%; more preferably, by more than 5%; more preferably, by more than 10%; and most preferably, by more than 25%.
  • a decrease in the mortality rate of a population of treated subjects may be measured by any reproducible means.
  • a decrease in the mortality rate of a population may be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment with an active compound.
  • a decrease in the mortality rate of a population may also be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment with an active compound.
  • Treating cancer can result in a decrease in tumor growth rate.
  • tumor growth rate is reduced by at least 5% relative to number prior to treatment; more preferably, tumor growth rate is reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%.
  • Tumor growth rate may be measured by any reproducible means of measurement. Tumor growth rate can be measured according to a change in tumor diameter per unit time.
  • Treating cancer can result in a decrease in tumor regrowth.
  • tumor regrowth is less than 5%; more preferably, tumor regrowth is less than 10%; more preferably, less than 20%; more preferably, less than 30%; more preferably, less than 40%; more preferably, less than 50%; even more preferably, less than 50%; and most preferably, less than 75%.
  • Tumor regrowth may be measured by any reproducible means of measurement. Tumor regrowth is measured, for example, by measuring an increase in the diameter of a tumor after a prior tumor shrinkage that followed treatment. A decrease in tumor regrowth is indicated by failure of tumors to reoccur after treatment has stopped.
  • Treating or preventing a cell proliferative disorder can result in a reduction in the rate of cellular proliferation.
  • the rate of cellular proliferation is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%.
  • the rate of cellular proliferation may be measured by any reproducible means of measurement.
  • the rate of cellular proliferation is measured, for example, by measuring the number of dividing cells in a tissue sample per unit time.
  • Treating or preventing a cell proliferative disorder can result in a reduction in the proportion of proliferating cells.
  • the proportion of proliferating cells is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%.
  • the proportion of proliferating cells may be measured by any reproducible means of measurement.
  • the proportion of proliferating cells is measured, for example, by quantifying the number of dividing cells relative to the number of nondividing cells in a tissue sample.
  • the proportion of proliferating cells can be equivalent to the mitotic index.
  • Treating or preventing a cell proliferative disorder can result in a decrease in size of an area or zone of cellular proliferation.
  • size of an area or zone of cellular proliferation is reduced by at least 5% relative to its size prior to treatment; more preferably, reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%.
  • Size of an area or zone of cellular proliferation may be measured by any reproducible means of measurement.
  • the size of an area or zone of cellular proliferation may be measured as a diameter or width of an area or zone of cellular proliferation.
  • Treating or preventing a cell proliferative disorder can result in a decrease in the number or proportion of cells having an abnormal appearance or morphology.
  • the number of cells having an abnormal morphology is reduced by at least 5% relative to its size prior to treatment; more preferably, reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%.
  • An abnormal cellular appearance or morphology may be measured by any reproducible means of measurement.
  • An abnormal cellular morphology can be measured by microscopy, e.g., using an inverted tissue culture microscope.
  • An abnormal cellular morphology can take the form of nuclear pleiomorphism.
  • the term "selectively" means tending to occur at a higher frequency in one population than in another population.
  • the compared populations can be cell populations.
  • a compound described herein i.e., the hydrobromide of Compound I, as well as Polymorph A
  • a compound described herein acts selectively to modulate one molecular target (e.g., a target protein methyltransferase) but does not significantly modulate another molecular target (e.g., a non-target protein methyltransferase).
  • the invention also provides a method for selectively inhibiting the activity of an enzyme, such as a protein methyltransferase.
  • an event occurs selectively in population A relative to population B if it occurs greater than two times more frequently in population A as compared to population B.
  • An event occurs selectively if it occurs greater than five times more frequently in population A.
  • An event occurs selectively if it occurs greater than ten times more frequently in population A; more preferably, greater than fifty times; even more preferably, greater than 100 times; and most preferably, greater than 1000 times more frequently in population A as compared to population B.
  • cell death would be said to occur selectively in cancer cells if it occurred greater than twice as frequently in cancer cells as compared to normal cells.
  • a compound described herein can modulate the activity of a molecular target (e.g., a target protein methyltransferase). Modulating refers to stimulating or inhibiting an activity of a molecular target.
  • a compound described herein modulates the activity of a molecular target if it stimulates or inhibits the activity of the molecular target by at least 2-fold relative to the activity of the molecular target under the same conditions but lacking only the presence of said compound.
  • a compound described herein modulates the activity of a molecular target if it stimulates or inhibits the activity of the molecular target by at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold relative to the activity of the molecular target under the same conditions but lacking only the presence of said compound.
  • the activity of a molecular target may be measured by any reproducible means.
  • the activity of a molecular target may be measured in vitro or in vivo.
  • the activity of a molecular target may be measured in vitro by an enzymatic activity assay or a DNA binding assay, or the activity of a molecular target may be measured in vivo by assaying for expression of a reporter gene.
  • a compound described herein i.e., the hydrobromide of Compound I, as well as Polymorph A
  • does not significantly modulate the activity of a molecular target if the addition of the compound does not stimulate or inhibit the activity of the molecular target by greater than 10% relative to the activity of the molecular target under the same conditions but lacking only the presence of said compound.
  • a compound described herein demonstrates a minimum of a fourfold differential, preferably a tenfold differential, more preferably a fifty fold differential, in the dosage required to achieve a biological effect.
  • a compound described herein demonstrates this differential across the range of inhibition, and the differential is exemplified at the IC 50 , i.e., a 50% inhibition, for a molecular target of interest.
  • Administering a compound described herein to a cell or a subject in need thereof can result in modulation (i.e., stimulation or inhibition) of an activity of a protein methyltransferase of interest.
  • Treating cancer or a cell proliferative disorder can result in cell death, and preferably, cell death results in a decrease of at least 10% in number of cells in a population. More preferably, cell death means a decrease of at least 20%; more preferably, a decrease of at least 30%; more preferably, a decrease of at least 40%; more preferably, a decrease of at least 50%; most preferably, a decrease of at least 75%.
  • Number of cells in a population may be measured by any reproducible means. A number of cells in a population can be measured by fluorescence activated cell sorting (FACS), immunofluorescence microscopy and light microscopy. Methods of measuring cell death are as shown in Li et al., Proc Natl Acad Sci U S A. 100(5): 2674-8, 2003 . In an aspect, cell death occurs by apoptosis.
  • an effective amount of a compound described herein is not significantly cytotoxic to normal cells.
  • a therapeutically effective amount of a compound is not significantly cytotoxic to normal cells if administration of the compound in a therapeutically effective amount does not induce cell death in greater than 10% of normal cells.
  • a therapeutically effective amount of a compound does not significantly affect the viability of normal cells if administration of the compound in a therapeutically effective amount does not induce cell death in greater than 10% of normal cells.
  • cell death occurs by apoptosis.
  • Contacting a cell with a compound described herein can induce or activate cell death selectively in cancer cells.
  • Administering to a subject in need thereof a compound described herein can induce or activate cell death selectively in cancer cells.
  • Contacting a cell with a compound described herein can induce cell death selectively in one or more cells affected by a cell proliferative disorder.
  • administering to a subject in need thereof a compound described herein induces cell death selectively in one or more cells affected by a cell proliferative disorder.
  • Described herein is a method of treating or preventing cancer (e.g, the course of which can be influenced by modulating EZH2-mediated protein methylation) by administering a compound of described herein (i.e., the hydrobromide of Compound I, as well as Polymorph A) to a subject in need thereof, where administration of the compound results in one or more of the following: prevention of cancer cell proliferation by accumulation of cells in one or more phases of the cell cycle (e.g.
  • G1, G1/S, G2/M induction of cell senescence, or promotion of tumor cell differentiation
  • therapeutic index is the maximum tolerated dose divided by the efficacious dose. Also described herein is a method used to identify suitable candidates for treating or preventing cancer.
  • a colorless plate crystal with dimensions 0.28 x 0.22 x 0.06 mm was mounted on a Nylon loop using very small amount of paratone oil.
  • Data were collected using a Bruker CCD (charge coupled device) based diffractometer equipped with an Oxford Cryostream low-temperature apparatus operating at 173 K. Data were measured using omega and phi scans of 0.5° per frame for 45 s. The total number of images was based on results from the program COSMO where redundancy was expected to be 4.0 and completeness to 100% out to 0.83 ⁇ .
  • Cell parameters were retrieved using APEX II software and refined using SAINT on all observed reflections. Data reduction was performed using the SAINT software which corrects for Lp.
  • DVS was measured on a VTI Model SGA-100 system. Measurement method: The relative humidity (RH) was changed in a controlled fashion, in 5% steps from 5.0% to 95.0% then back to 5.0% using the gravimetric vapor sorption system, and the weight percentage change (wt%) of the sample at each stage was measured.
  • RH relative humidity
  • wt% weight percentage change
  • HPLC HPLC was conducted on an Agilent 1200 HPLC quaternary pump, low pressure mixing, with an in-line degasser.
  • Analytical method conditions 8 ⁇ L sample (20 mg of ER-581982-06 diluted with 50 mL of a methanol to provide approximately 0.4 mg/mL solution) was injected onto a Agilent Zorbax Eclipse XDB-C18 (4.6 x 150 mm, 3.5 um), Chromatography conditions: mobile phase A, water with 5mM ammonium formate; mobile phase B, 5 mM ammonium formate in 50/45/5 acetonitrile/methanol/water; flow rate, 1.5 ml/min.; gradient: isocratic at 10% B from 0 to 3 min; linear increase to 70% B from 3 to 7 min; isocratic at 70% B from 7 to 12 min; linear increase to 100% B from 12 to 15 min isocratic at 100% B from 15 to 20 min; column temperature, 35 °C; detection, UV 230 nm
  • reaction mixture was stirred at room temperature for 3 hours. Upon completion of the reaction as determined by TLC, aqueous sodium bicarbonate solution was added to the reaction mixture until a pH 7-8 was obtained, the organic phase was separated and the aqueous phase was extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by column chromatography (100-200 mesh silica gel) eluting with ethyl acetate: hexane to afford the desired compound as a viscous liquid (14 g, 93%).
  • the mixture was stirred at 40°C for 18 h. After cooling to 0-5 °C, the mixture was neutralized to pH 6.5 with 1 N hydrochloric acid (580 mL) maintaining the temperature below 25 °C. Then, the mixture was extracted twice with a mixture of dichloromethane (690 mL) and methanol (69.0 mL). The organic layers were combined and concentrated under vacuum to give a crude product as a yellow solid (127g).
  • hydrobromide (HBr) salt had the most advantageous physicochemical properties in terms of ease of preparation and hygroscopicity.
  • the monohydrobromide was the most crystalline and least hydroscopic (see Figures 1 , 3 , and 4 ). Furthermore, the monohydrobromide is highly stable, and resists generation of impurities ( Figure 5 depicts HPLC analysis of Polymorph A over three days at an elevated temperature. Polymorph A produced minimal impurities over three days 100°C). Interestingly, the di-HBr salt of Compound I was found to be primarily amorphous ( Figure 2 ).
  • Polymorphs A and B Two different crystal forms of the monohydrobromide of Compound I (Polymorphs A and B) were obtained from different solvent systems and characterized using XRD, DSC and TGA-DSC analyses. XRD and DSC data for these two different crystal forms from representative batches of Compound I are shown in Figure 1 , Figure 3 , and Figure 10 .
  • Polymorph B is characterized by a powder XRD pattern with peaks at 8.5, 10.9, 16.7, 17.4, 20.9, 22.1 and 25.7 ⁇ 0.2 degrees 2 theta (see Figure 10 ). Between these two, Polymorph A was found to be more crystalline in nature. Dynamic vapor adsorption (DVS) studies showed that the polymorph A is non-hygroscopic ( Figure 4 ). In the thermal analyses, a single endothermic peak was observed with an onset temperature approximately at 251 °C. In addition, it was evident from DSC analysis that the recrystallization of polymorph A significantly increases the crystallinity of the material (see Figure 3
  • SAM S -adenosylmethionine
  • SAH S -adenosylhomocyteine
  • bicine KCl
  • Tween20 dimethylsulfoxide (DMSO)
  • BSG bovine skin gelatin
  • DTT Dithiothreitol
  • EMD EMD
  • 3 H-SAM was purchased from American Radiolabeled Chemicals with a specific activity of 80 Ci/mmol.
  • 384-well streptavidin Flashplates were purchased from PerkinElmer.
  • Chicken erythrocyte oligonucleosomes were purified from chicken blood according to established procedures.
  • PRC2 Complexes Human PRC2 complexes were purified as 4-component enzyme complexes co-expressed in Spodoptera frugiperda (sf9) cells using a baculovirus expression system. The subunits expressed were wild-type EZH2 (NM_004456) or EZH2 Y641F, N, H, S or C mutants generated from the wild-type EZH2 construct, EED (NM_003797), Suz12 (NM_015355) and RbAp48 (NM_005610). The EED subunit contained an N-terminal FLAG tag that was used to purify the entire 4-component complex from sf9 cell lysates.
  • the purity of the complexes met or exceeded 95% as determined by SDS-PAGE and Agilent Bioanalyzer analysis. Concentrations of enzyme stock concentrations (generally 0.3 - 1.0 mg/mL) was determined using a Bradford assay against a bovine serum albumin (BSA) standard.
  • BSA bovine serum albumin
  • DMSO DMSO (1 ⁇ L) was added to columns 11, 12, 23, 24, rows A - H for the maximum signal control, and SAH, a known product and inhibitor of PRC2 (1 ⁇ L) was added to columns 11,12, 23, 24, rows I - P for the minimum signal control.
  • a cocktail (40 ⁇ L) containing the wild-type PRC2 enzyme and H3K27me0 peptide or any of the Y641 mutant enzymes and H3K27me2 peptide was added by Multidrop Combi (Thermo). The compounds were allowed to incubate with PRC2 for 30 min at 25 °C, then a cocktail (10 ⁇ L) containing a mixture of non-radioactive and 3 H-SAM was added to initiate the reaction (final volume 51 ⁇ L).
  • the final concentrations were as follows: wild-type or mutant PRC2 enzyme was 4 nM, SAH in the minimum signal control wells was 1 mM and the DMSO concentration was 1%. The final concentrations of the rest of the components are indicated in Table 2, below.
  • the assays were stopped by the addition of non-radioactive SAM (10 ⁇ L) to a final concentration of 600 ⁇ M, which dilutes the 3 H-SAM to a level where its incorporation into the peptide substrate is no longer detectable.
  • Table 2 Final concentrations of components for each assay variation based upon EZH2 identity (wild-type or Y641 mutant EZH2) PRC2 Enzyme (denoted by EZH2 identity) Peptide (nM) Non-radioactive SAM (nM) 3 H-SAM (nM) Wild-type 185 1800 150 Y641F 200 850 150 Y641N 200 850 150 Y641H 200 1750 250 Y641S 200 1300 200 Y641C 200 3750 250
  • DMSO DMSO (1 ⁇ L) was added to columns 11, 12, 23, 24, rows A - H for the maximum signal control, and SAH, a known product and inhibitor of PRC2 (1 ⁇ L) was added to columns 11,12, 23, 24, rows I - P for the minimum signal control.
  • a cocktail (40 ⁇ L) containing the wild-type PRC2 enzyme and chicken erythrocyte oligonucleosome was added by Multidrop Combi (Thermo). The compounds were allowed to incubate with PRC2 for 30 min at 25 °C, then a cocktail (10 ⁇ L) containing a mixture of non-radioactive and 3 H-SAM was added to initiate the reaction (final volume 51 ⁇ L).
  • the final concentrations were as follows: wild-type PRC2 enzyme was 4 nM, non-radioactive SAM was 430 nM, 3 H-SAM was 120 nM, chicken erythrocyte olumbleucleosome was 120 nM, SAH in the minimum signal control wells was 1 mM and the DMSO concentration was 1%.
  • the assay was stopped by the addition of non-radioactive SAM (10 ⁇ L) to a final concentration of 600 ⁇ M, which dilutes the 3 H-SAM to a level where its incorporation into the chicken erythrocyte olignonucleosome substrate is no longer detectable.
  • top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit.
  • the Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit.
  • Y is the % inhibition and X is the compound concentration.
  • IC 50 values for the PRC2 enzyme assays on peptide substrates are presented in Table 3 below.
  • WSU-DLCL2 suspension cells were purchased from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany).
  • RPMI/Glutamax Medium, Penicillin-Streptomycin, Heat Inactivated Fetal Bovine Serum, and D-PBS were purchased from Life Technologies, Grand Island, NY, USA.
  • Extraction Buffer and Neutralization Buffer(5X) were purchased from Active Motif, Carlsbad, CA, USA.
  • Rabbit anti-Histone H3 antibody was purchased from Abcam, Cambridge, MA, USA.
  • Rabbit anti-H3K27me3 and HRP-conjugated anti-rabbit-IgG were purchased from Cell Signaling Technology, Danvers, MA, USA.
  • TMB "Super Sensitive" substrate was sourced from BioFX Laboratories, Owings Mills, MD, USA.
  • IgG-free Bovine Serum Albumin was purchased from Jackson ImmunoResearch, West Grove, PA, USA.
  • PBS with Tween (10X PBST) was purchased from KPL, Gaithersburg, MD, USA.
  • Sulfuric Acid was purchased from Ricca Chemical, Arlington, TX, USA.
  • Immulon ELISA plates were purchased from Thermo, Rochester, NY, USA.
  • V-bottom cell culture plates were purchased from Corning Inc., Corning, NY, USA.V-bottom polypropylene plates were purchased from Greiner Bio-One, Monroe, NC, USA.
  • WSU-DLCL2 suspension cells were maintained in growth medium (RPMI 1640 supplemented with 10% v/v heat inactivated fetal bovine serum and 100 units/mL penicillin-streptomycin) and cultured at 37°C under 5% CO 2 . Under assay conditions, cells were incubated in Assay Medium (RPMI 1640 supplemented with 20% v/v heat inactivated fetal bovine serum and 100 units/mL penicillin-streptomycin) at 37°C under 5% CO 2 on a plate shaker.
  • growth medium RPMI 1640 supplemented with 10% v/v heat inactivated fetal bovine serum and 100 units/mL penicillin-streptomycin
  • WSU-DLCL2 cells were seeded in assay medium at a concentration of 50,000 cells per mL to a 96-well V-bottom cell culture plate with 200 ⁇ L per well.
  • Compound (1 ⁇ L) from 96 well source plates was added directly to V-bottom cell plate. Plates were incubated on a titer-plate shaker at 37°C, 5% CO2 for 96 hours. After four days of incubation, plates were spun at 241 x g for five minutes and medium was aspirated gently from each well of cell plate without disturbing cell pellet. Pellet was resuspended in 200 ⁇ L DPBS and plates were spun again at 241 x g for five minutes.
  • Histone H3 detection plate 100 ⁇ L per well were added of anti-Histone-H3 antibody (Abcam, ab1791) diluted 1:10,000 in ELISA Diluent.
  • H3K27 trimethylation detection plate 100 ⁇ L per well were added of anti-H3K27me3 diluted 1:2000 in ELISA diluent. Plates were incubated for 90 minutes at room temperature. Plates were washed three times with 300 ⁇ L 1X PBST per well.
  • HRP-conjugated anti-rabbit IgG antibody diluted to 1:6000 in ELISA diluent was added per well.
  • H3K27me3 detection 100 ⁇ L of HRP conjugated anti-rabbit IgG antibody diluted to 1:4000 in ELISA diluent was added per well. Plates were incubated at room temperature for 90 minutes. Plates were washed four times with 1X PBST 300 ⁇ L per well. TMB substrate100 ⁇ L was added per well. Histone H3 plates were incubated for five minutes at room temperature. H3K27me3 plates were incubated for 10 minutes at room temperature. The reaction was stopped with sulfuric acid IN (100 ⁇ L per well). Absorbance for each plate was read at 450 nm.
  • Each plate included eight control wells of DMSO only treatment (Minimum Inhibition) as well as eight control wells for maximum inhibition (Background wells).
  • WSU-DLCL2 suspension cells were purchased from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany).
  • RPMI/Glutamax Medium, Penicillin-Streptomycin, Heat Inactivated Fetal Bovine Serum were purchased from Life Technologies, Grand Island, NY, USA.
  • V-bottom polypropylene 384-well plates were purchased from Greiner Bio-One, Monroe, NC, USA.
  • Cell culture 384-well white opaque plates were purchased from Perkin Elmer, Waltham, MA, USA.
  • Cell-Titer Glo® was purchased from Promega Corporation, Madison, WI, USA.
  • SpectraMax M5 plate reader was purchased from Molecular Devices LLC, Sunnyvale, CA, USA.
  • WSU-DLCL2 suspension cells were maintained in growth medium (RPMI 1640 supplemented with 10% v/v heat inactivated fetal bovine serum and cultured at 37°C under 5% CO2. Under assay conditions, cells were incubated in Assay Medium (RPMI 1640 supplemented with 20% v/v heat inactivated fetal bovine serum and 100 units/mL penicillin-streptomycin) at 37°C under 5% CO2.
  • mice Female Fox Chase SCID® Mice (CB 17/Icr- Prkdcscid / I crIcoCrl, Beijing Vitalriver Laboratory Animal Co., LTD) were 6 - 8 weeks old and had a body-weight (BW) range of 16.0-21.1 g on D1 of the study.
  • the animals were fed ad libitum with water (sterile) and irradiation sterilized dry granule food.
  • the mice were housed on corn cob bedding in static microisolators on a 12-hour light cycle at 20-22 °C (68-72 °F) and 40-60% humidity. All procedures comply with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care.
  • Human lymphoma cell line SUDHL10 was obtained from DSMZ and maintained at the CRO as suspension cultures in RPMI-1640 medium containing 100 units/mL penicillin G sodium salt, 100 g/mL streptomycin and 10% fetal bovine serum. The cells were cultured in tissue culture flasks in a humidified incubator at 37 °C, in an atmosphere of 5% CO2 and 95% air. Only cultures below passage 12 were used for implantation
  • the hydrobromide of Compound I was stored at room temperature and protected from light. On each treatment day, a fresh compound formulations were prepared by suspending the powder in 0.5% sodium carboxymethylcellulose (NaCMC) and 0.1% Tween® 80 in deionized water. The vehicle, 0.5% NaCMC and 0.1% Tween® 80 in deionized water, was used to treat the control group at the same schedule. Formulations were stored away from light at 4 °C prior to administration.
  • NaCMC sodium carboxymethylcellulose
  • Tween® 80 0.1%
  • mice were treated with doses of the hydrobromide of Compound I ranging from 125 - 500 mg/kg and at a BID (twice a day every 12 h) schedules for 28 days by oral gavage. Each dose was delivered in a volume of 0.2 mL/20 g mouse (10 mL/kg), and adjusted for the last recorded weight of individual animals. On day 25 the 8 mice with the smallest tumors per group were chosen for a tumor growth delay endpoint (observation up to 60 days). The remaining animals were euthanized on day 28 3h after the last dose for tumor collection.
  • MTV Median Tumor Volume
  • TGI Tumor Growth Inhibition
  • MTV(n) the median tumor volume for the number of animals, n, evaluable on the last day, was determined for each group.
  • mice per group were kept alive after the last treatment day for tumor growth delay analysis. Tumors were callipered twice-weekly and each test animal was euthanized when its neoplasm reached the endpoint volume of 2000 mm 3 or on the pre-specified last day of the study, whichever came first. Kaplan Meier survival analysis was performed.
  • mice were weighed daily on Days 1-5, and then twice weekly until the completion of the study. The mice were examined frequently for overt signs of any adverse, treatment related side effects, which were documented. Acceptable toxicity for the maximum tolerated dose (MTD) was defined as a group mean BW loss of less than 20% during the test, and not more than 10% mortality due to TR deaths. A death was to be classified as TR if it was attributable to treatment side effects as evidenced by clinical signs and/or necropsy, or due to unknown causes during the dosing period. A death was to be classified as NTR if there was evidence that the death was unrelated to treatment side effects.
  • MTD maximum tolerated dose
  • NTR deaths during the dosing interval would typically be categorized as NTRa (due to an accident or human error) or NTRm (due to necropsy-confirmed tumor dissemination by invasion and/or metastasis). Orally treated animals that die from unknown causes during the dosing period may be classified as NTRu when group performance does not support a TR classification and necropsy, to rule out a dosing error, is not feasible.
  • mice with the largest tumors were sampled in a pre-specified fashion to assess target inhibition in tumors.
  • Tumors were harvested from specified mice under RNAse free conditions and bisected. Total tumor weight was measured. Frozen tumor tissue from each animal was snap frozen in liquid N 2 and pulverized with a mortar and pestle.
  • tumor tissue was homogenized in 1.5 ml nuclear extraction buffer (10 mM Tris-HCl, 10 mM MgCl2, 25 mM KCl, 1% Triton X-100, 8.6% Sucrose, plus a Roche protease inhibitor tablet 1836145) and incubated on ice for 5 minutes. Nuclei were collected by centrifugation at 600 g for 5 minutes at 4° C and washed once in PBS. Supernatant was removed and histones extracted for one hour, with vortexing every 15 minutes, with 0.4 N cold sulfuric acid.
  • nuclear extraction buffer 10 mM Tris-HCl, 10 mM MgCl2, 25 mM KCl, 1% Triton X-100, 8.6% Sucrose, plus a Roche protease inhibitor tablet 1836145
  • Extracts were clarified by centrifugation at 10000 g for 10 minutes at 4°C and transferred to a fresh microcentrifuge tube containing 10x volume of ice cold acetone. Histones were precipitated at -20° C for 2 hours-overnight, pelleted by centrifugation at 10000 g for 10 minutes and resuspended in water.
  • Histones were prepared in equivalent concentrations in coating buffer (PBS+0.05%BSA) yielding 0.5 ng/ul of sample, and 100 ul of sample or standard was added in duplicate to 2 96-well ELISA plates (Thermo Labsystems, Immulon 4HBX #3885). The plates were sealed and incubated overnight at 4°C. The following day, plates were washed 3x with 300 ul/well PBST (PBS+0.05% Tween 20; 10X PBST, KPL #51-14-02) on a Bio Tek plate washer.
  • coating buffer PBS+0.05%BSA
  • Plates were blocked with 300 ul/well of diluent (PBS+2%BSA+0.05% Tween 20), incubated at RT for 2 hours, and washed 3x with PBST. All antibodies were diluted in diluent. 100 ul/well of anti-H3K27me3 (CST #9733, 50% glycerol stock 1:1,000) or anti-total H3 (Abcam ab1791, 50% glycerol 1:10,000) was added to each plate. Plates were incubated for 90 min at RT and washed 3x with PBST.
  • CST #9733 50% glycerol stock 1:1,000
  • Anti-total H3 Abcam ab1791, 50% glycerol 1:10,000
  • mice bearing SUDHL10 tumor xenografts were treated with the hydrobromide of Compound I at the maximal tolerated dose of 500 mg/kg BID and fractions of the MTD (1 ⁇ 2 and 1 ⁇ 4 MTD). All doses were well tolerated for 28 days without any significant body weight loss. There was one non-treatment related death in the 500 mg/kg group on day 15 due to a dosing error. All doses resulted in tumor growth inhibition when compared to vehicle on day 28 (Table 4), and the 250 mg/kg and 500 mg/kg BID groups induced regressions (TGI > 100%).
  • Table 4 Summary of tumor growth inhibition values induced by hydrobromide of Compound I in SUDHL10 xenografts Group % TGI from day 1 % TGI from day 8 125 mg/kg BID 54 57 250 mg/kg BID 101 113 500 mg/kg BID 104 115
  • Figure 12A shows the growth of the SUDHL10 xenograft tumors over time for the different treatment groups.
  • the 125 mg/kg BID group was not significantly different from the vehicle group by repeated measures ANOVA and Dunnett's post test, but the mean terminal tumor size on day 28 was significantly smaller than the one in the vehicle group (2 way ANOVA with Bonferroni post test, p ⁇ 0.0001).
  • Dosing of 250 mg/kg BID and 500 mg/kg BID of the hydrobromide of Compound I for 28 days induced comparable regression responses as the terminal tumor weights on day 28 were similar for those 2 groups ( Figure 12B ).
  • FIG. 13 shows a clear dose dependent down-regulation of the H3K27me3 methyl mark with treatment by the hydrobromide of Compound I.
  • This figure shows global H3K27me3 methylation in SUDHL10 tumors from mice treated with the hydrobromide of Compound I for 28 days.
  • the monohydrobromide of Compound I was tested for its anti-cancer activity in Pfeiffer mouse xenograft model, which is a human diffused large B-Cell lymphoma xenograft model.
  • Pfeiffer mouse xenograft model which is a human diffused large B-Cell lymphoma xenograft model.
  • Female of 5-week old NSG mice (Jackson Labs, Bar Harbor, Maine) were implanted subcutaneously with 20 to 25 mg tumor fragments. Treatment was started approximately 31 days after the tumor implantation, when the average tumors reached approximately 365 mm 3 . The treatment scheme is described in Table 5. Table 5.
  • Day 1 was the day of the first treatment, and Day 28 was the day of the last treatment. This study was terminated 36 days after the last dose, so Day 64 was the day of study termination.
  • the primary endpoints used to evaluate efficacy in this study were complete tumor regressions (CR), tumor sizes among groups, and percentage of tumor inhibition at the end of the study. A complete response was defined as a decrease in tumor size to an undetectable size ( ⁇ 20mm 3 ) at the end of the study. Values for percentage of tumor inhibition were calculated from the formula [1-( ⁇ T/ ⁇ C)] x 100, where ⁇ T and ⁇ C are changes in mean tumor volume ( ⁇ growth) for each treated (T) and vehicle control group (C).
  • T 0 and C 0 (one day before the first dose) were used for the starting tumor volume. Additionally, tumor volumes which were taken one day after the last dose (T 29 and C 29 ) were used for the calculation of ⁇ T and ⁇ C. When the value was more than 100%, it was concluded as 100%.
  • the hydrobromide of Compound I showed potent and long-lasting anti-cancer activity in Pfeiffer model with 100% of CR rate in three out of four dosing group (Table 6). Moreover, tumor re-growth was not observed even 36 days after cessation of the treatment. This suggests that all the tumor cells were killed during the treatment. Although tumor re-growth was observed in the group with the lowest dose (group B, 34.2 mg/kg), clear tumor stasis activity was observed during the treatment period ( Figure 16 ). Tumor only started to grow upon cessation of the treatment ( Figure 16 ). This result also suggests that the tumor stasis activity observed in group B is indeed test article-induced activity. Table 6.

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Claims (23)

  1. Bromhydrate de N-((4,6-diméthyl-2-oxo-1,2-dihydropyridin-3-yl)méthyl)-5-(éthyl(tétrahydro-2H-pyran-4-yl)amino)-4-méthyl-4'-(morpholinométhyl)-[1,1'-biphényl]-3-carboxamide destiné à être utilisé dans le traitement d'un trouble prolifératif cellulaire du système hématologique.
  2. Composé destiné à être utilisé selon la revendication 1, qui est un monobromhydrate.
  3. Composé destiné à être utilisé selon l'une quelconque des revendications 1 et 2, ledit composé étant cristallin.
  4. Composé destiné à être utilisé selon l'une quelconque des revendications précédentes, ledit composé étant exempt d'impuretés.
  5. Composé destiné à être utilisé selon l'une quelconque des revendications précédentes, ledit composé étant un solide cristallin sensiblement exempt de bromhydrate de N-((4,6-diméthyl-2-oxo-1,2-dihydropyridin-3-yl)méthyl)-5-(éthyl(tétrahydro-2H-pyran-4-yl)amino)-4-méthyl-4'-(morpholinométhyl)-[1,1'-biphényl]-3-carboxamide amorphe.
  6. Polymorphe A de bromhydrate de N-((4,6-diméthyl-2-oxo-1,2-dihydropyridin-3-yl)méthyl)-5-(éthyl(tétrahydro-2H-pyran-4-yl)amino)-4-méthyl-4'-(morpholinométhyl)-[1,1'-biphényl]-3-carboxamide destiné à être utilisé dans le traitement d'un trouble prolifératif cellulaire du système hématologique ; le Polymorphe A présentant un diagramme de diffraction des rayons X sur poudre ayant un ou plusieurs pics caractéristiques exprimés en degrés 2-thêta à 3,9 +/- 0,3 degrés, 17,5 +/- 0,3 degrés et 22,0 +/- 0,3 degrés 2-thêta.
  7. Polymorphe destiné à être utilisé selon la revendication 7 ; le polymorphe présentant un diagramme de diffraction des rayons X sur poudre ayant des pics caractéristiques exprimés en degrés 2-thêta à 3,9 +/- 0,3 degrés, 17,5 +/- 0,3 degrés et 22,0 +/- 0,3 degrés 2-thêta.
  8. Polymorphe destiné à être utilisé selon la revendication 7 ; le polymorphe présentant un diagramme de diffraction des rayons X sur poudre ayant un ou plusieurs pics caractéristiques exprimés en degrés 2-thêta à 3,9 +/- 0,3 degrés, 14,3 +/- 0,3 degrés, 18,7 +/- 0,3 degrés, 23,3 +/- 0,3 degrés et environ 23,6 +/- 0,3 degrés 2-thêta.
  9. Polymorphe destiné à être utilisé selon la revendication 7 ; le polymorphe présentant un diagramme de diffraction des rayons X sur poudre ayant au moins 5 pics caractéristiques exprimés en degrés 2-thêta à 3,9 +/- 0,3 degrés, 10,1 +/- 0,3 degrés, 14,3 +/- 0,3 degrés, 17,5 +/- 0,3 degrés, 18,7 +/- 0,3 degrés, 20,6 +/- 0,3 degrés, 20,9 +/- 0,3 degrés, 21,8 +/- 0,3 degrés, 22,0 +/- 0,3 degrés, 23,3 +/- 0,3 degrés et 23,6 +/- 0,3 degrés 2-thêta.
  10. Polymorphe destiné à être utilisé selon la revendication 7 ; le polymorphe présentant un diagramme de diffraction des rayons X sur poudre ayant au moins 6 pics caractéristiques exprimés en degrés 2-thêta à 3,9 +/- 0,3 degrés, 10,1 +/- 0,3 degrés, 14,3 +/- 0,3 degrés, 17,5 +/- 0,3 degrés, 18,7 +/- 0,3 degrés, 20,6 +/- 0,3 degrés, 20,9 +/- 0,3 degrés, 21,8 +/- 0,3 degrés, 22,0 +/- 0,3 degrés, 23,3 +/- 0,3 degrés et 23,6 +/- 0,3 degrés 2-thêta.
  11. Polymorphe destiné à être utilisé selon la revendication 7 ; le polymorphe présentant un diagramme de diffraction des rayons X sur poudre ayant au moins 7 pics caractéristiques exprimés en degrés 2-thêta à 3,9 +/- 0,3 degrés, 10,1 +/- 0,3 degrés, 14,3 +/- 0,3 degrés, 17,5 +/- 0,3 degrés, 18,7 +/- 0,3 degrés, 20,6 +/- 0,3 degrés, 20,9 +/- 0,3 degrés, 21,8 +/- 0,3 degrés, 22,0 +/- 0,3 degrés, 23,3 +/- 0,3 degrés et 23,6 +/- 0,3 degrés 2-thêta.
  12. Polymorphe destiné à être utilisé selon la revendication 7 ; le polymorphe présentant un diagramme de diffraction des rayons X sur poudre ayant au moins 8 pics caractéristiques exprimés en degrés 2-thêta à 3,9 +/- 0,3 degrés, 10,1 +/- 0,3 degrés, 14,3 +/- 0,3 degrés, 17,5 +/- 0,3 degrés, 18,7 +/- 0,3 degrés, 20,6 +/- 0,3 degrés, 20,9 +/- 0,3 degrés, 21,8 +/- 0,3 degrés, 22,0 +/- 0,3 degrés, 23,3 +/- 0,3 degrés et 23,6 +/- 0,3 degrés 2-thêta.
  13. Polymorphe destiné à être utilisé selon la revendication 7 ; le polymorphe présentant un diagramme de diffraction des rayons X sur poudre ayant au moins 9 pics caractéristiques exprimés en degrés 2-thêta à 3,9 +/- 0,3 degrés, 10,1 +/- 0,3 degrés, 14,3 +/- 0,3 degrés, 17,5 +/- 0,3 degrés, 18,7 +/- 0,3 degrés, 20,6 +/- 0,3 degrés, 20,9 +/- 0,3 degrés, 21,8 +/- 0,3 degrés, 22,0 +/- 0,3 degrés, 23,3 +/- 0,3 degrés et 23,6 +/- 0,3 degrés 2-thêta.
  14. Polymorphe destiné à être utilisé selon la revendication 7 ; le polymorphe présentant un diagramme de diffraction des rayons X sur poudre ayant au moins 10 pics caractéristiques exprimés en degrés 2-thêta à 3,9 +/- 0,3 degrés, 10,1 +/- 0,3 degrés, 14,3 +/- 0,3 degrés, 17,5 +/- 0,3 degrés, 18,7 +/- 0,3 degrés, 20,6 +/- 0,3 degrés, 20,9 +/- 0,3 degrés, 21,8 +/- 0,3 degrés, 22,0 +/- 0,3 degrés, 23,3 +/- 0,3 degrés et 23,6 +/- 0,3 degrés 2-thêta.
  15. Polymorphe destiné à être utilisé selon la revendication 7 ; le polymorphe présentant un diagramme de diffraction des rayons X sur poudre ayant des pics caractéristiques exprimés en degrés 2-thêta à 3,9 +/- 0,3 degrés, 10,1 +/- 0,3 degrés, 14,3 +/- 0,3 degrés, 17,5 +/- 0,3 degrés, 18,7 +/- 0,3 degrés, 20,6 +/- 0,3 degrés, 20,9 +/- 0,3 degrés, 21,8 +/- 0,3 degrés, 22,0 +/- 0,3 degrés, 23,3 +/- 0,3 degrés et 23,6 +/- 0,3 degrés 2-thêta.
  16. Polymorphe destiné à être utilisé selon l'une quelconque des revendications 6 à 15, le polymorphe présentant un diagramme de diffraction des rayons X sur poudre sensiblement conforme à la figure 1 et/ou le polymorphe présentant un diagramme de diffraction des rayons X sur poudre sensiblement conforme au tableau 1 : Tableau 1 Pics (Degrés 2-thêta) 3,9 10,1 14,3 17,5 18,7 20,6 20,9 21,8 22,0 23,3 23,6
  17. Polymorphe destiné à être utilisé selon l'une quelconque des revendications 6 à 16, le polymorphe présentant un thermogramme de calorimétrie différentielle à balayage ayant un pic caractéristique exprimé en unités de °C à une température de 255 +/- 5°C.
  18. Polymorphe destiné à être utilisé selon l'une quelconque des revendications 6 à 17, le polymorphe présentant un thermogramme de calorimétrie différentielle à balayage sensiblement conforme à la figure 3.
  19. Polymorphe destiné à être utilisé selon l'une quelconque des revendications précédentes, le trouble prolifératif cellulaire du système hématologique étant un cancer hématologique.
  20. Polymorphe destiné à être utilisé selon la revendication 19, le cancer hématologique étant choisi parmi le myélome multiple, le lymphome, la leucémie, les néoplasies myéloïdes et les néoplasies mastocytaires.
  21. Polymorphe destiné à être utilisé selon la revendication 20, le lymphome étant choisi parmi le lymphome de Hodgkin, les lymphomes infantiles et les lymphomes d'origine lymphocytaire et cutanée.
  22. Polymorphe destiné à être utilisé selon la revendication 20, la leucémie étant choisie parmi la leucémie infantile, la leucémie à tricholeucocytes, la leucémie lymphocytaire aiguë, la leucémie myélocytaire aiguë, la leucémie lymphocytaire chronique, la leucémie myélocytaire chronique, la leucémie myélogène chronique et la leucémie à mastocytes.
  23. Polymorphe destiné à être utilisé selon la revendication 20, le cancer hématologique étant le myélome multiple.
EP16191716.6A 2012-04-13 2013-04-11 Bromhydrate de n-((4,6-diméthyl-2-oxo-l,2-dihydropyridin-3-yl)méthyl)-5-(éthyl (tétrahydro-2h-pyran-4-yl)amino)-4-méthyl-4'-(morpholinométhyl)-[l,1'-biphényl]-3-carboxamide pour l'utilisation dans le traitement d'un trouble de prolifération cellulaire du système hématologique Active EP3184523B1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
EP19180479.8A EP3628670B1 (fr) 2012-04-13 2013-04-11 Forme saline pour inhibition de ezh2
SI201331566T SI3184523T1 (sl) 2012-04-13 2013-04-11 N-((4,6 dimetil-2-okso-L,2-dihidropiridin-3-IL)metil)-5-(etil (tetrahidro-2H-piran-4-IL)amino)-4-metil-4'-(morfolinometil)-(L, 1'- bifen IL)-3-karboksamid hidrobromid za uporabo pri zdravljenju celične proliferativne motnje hematološkega sistema
DK19180479.8T DK3628670T3 (da) 2012-04-13 2013-04-11 Saltform til ezh2-hæmning
RSP20191175 RS59392B1 (sr) 2012-04-13 2013-04-11 N-((4,6-dimetil-2-okso-l,2-dihidropiridin-3-il)metil)-5-(etil(tetrahidro-2h-piran-4-il)amino)-4-metil-4’ (morfolinometil)-[l,1’-bifenil]-3-karboksamid hidrobromid za upotrebu u lečenju proliferativnih poremećaja ćelija hematološkog sistema
EP22200677.7A EP4190777A1 (fr) 2012-04-13 2013-04-11 Hbr forme saline pour inhibition de ezh2
PL16191716T PL3184523T3 (pl) 2012-04-13 2013-04-11 Bromowodorek N-((4,6-dimetylo-2-okso-1,2-dihydropirydyn-3-ylo)metylo)-5- (etylo(tetrahydro-2H-piran-4-ylo)amino)-4-metylo-4'-(morfolinometylo)-[1,1'- bifenylo]-3-karboksyamidu do zastosowania w leczeniu zaburzenia proliferacji komórkowej układu hematologicznego
HRP20191653TT HRP20191653T1 (hr) 2012-04-13 2019-09-13 N-((4,6-dimetil-2-okso-l,2-dihidropiridin-3-il)metil)-5-(etil(tetrahidro-2h-piran-4-il)amino)-4-metil-4' (morfolinometil)-[l,1'-bifenil]-3-karboksamid hidrobromid za uporabu u liječenju proliferativnih poremećaja stanica hematološkog sustava

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Families Citing this family (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JO3438B1 (ar) 2011-04-13 2019-10-20 Epizyme Inc مركبات بنزين مستبدلة بأريل أو أريل غير متجانس
EP2780013A4 (fr) 2011-11-18 2015-07-01 Constellation Pharmaceuticals Inc Modulateurs d'enzymes de modification par méthylation, compositions et utilisations associées
US9085583B2 (en) 2012-02-10 2015-07-21 Constellation—Pharmaceuticals, Inc. Modulators of methyl modifying enzymes, compositions and uses thereof
RU2019126908A (ru) 2012-04-13 2020-01-13 Эпизайм, Инк. Комбинированная терапия для лечения рака
HUE060881T2 (hu) 2012-04-13 2023-04-28 Epizyme Inc Sóforma az EZH2 gátláshoz
US9562041B2 (en) 2012-05-16 2017-02-07 Glaxosmithkline Llc Enhancer of zeste homolog 2 inhibitors
CA2887243C (fr) * 2012-10-15 2024-04-09 Epizyme, Inc. Methodes de traitement du cancer
EP2906537B1 (fr) 2012-10-15 2020-03-11 Epizyme, Inc. Composés de benzène substitué
US9745305B2 (en) 2013-03-15 2017-08-29 Constellation Pharmaceuticals, Inc. Modulators of methyl modifying enzymes, compositions and uses thereof
KR20160003115A (ko) 2013-04-30 2016-01-08 글락소스미스클라인 인털렉츄얼 프로퍼티 (넘버 2) 리미티드 제스트 인핸서 상동체 2 억제제
JP2016523955A (ja) 2013-07-10 2016-08-12 グラクソスミスクライン、インテレクチュアル、プロパティー、ナンバー２、リミテッドＧｌａｘｏｓｍｉｔｈｋｌｉｎｅ Ｉｎｔｅｌｌｅｃｔｕａｌ Ｐｒｏｐｅｒｔｙ Ｎｏ．２ Ｌｉｍｉｔｅｄ Ｚｅｓｔｅホモログ２エンハンサー阻害剤
US9969716B2 (en) 2013-08-15 2018-05-15 Constellation Pharmaceuticals, Inc. Indole derivatives as modulators of methyl modifying enzymes, compositions and uses thereof
PL3057962T3 (pl) 2013-10-16 2024-01-29 Epizyme, Inc. Postać soli chlorowodorku do inhibicji ezh2
AU2014337121A1 (en) * 2013-10-18 2016-04-14 Epizyme, Inc. Method of treating cancer
WO2015077193A1 (fr) * 2013-11-19 2015-05-28 Bristol-Myers Squibb Company Inhibiteurs de lysine méthyle transférase
KR102473113B1 (ko) 2013-12-06 2022-12-01 에피자임, 인코포레이티드 암 치료를 위한 병용 요법
SG10201811128RA (en) 2014-06-17 2019-01-30 Epizyme Inc Ezh2 inhibitors for treating lymphoma
EP3157928B1 (fr) 2014-06-20 2019-02-13 Constellation Pharmaceuticals, Inc. Formes cristallines de l'acétamide 2-((4s)-6-(4-chlorophényl)-1-méthyl-4 h-benzo[c]isoxazolo[4,5-e]azépin-4-yle)
NZ730365A (en) 2014-10-16 2024-03-22 Epizyme Inc Method for treating cancer
MX2017006089A (es) * 2014-11-17 2017-12-11 Epizyme Inc Metodo para tratar el cancer.
AR102767A1 (es) 2014-12-05 2017-03-22 Lilly Co Eli Inhibidores de ezh2
AU2016252546A1 (en) 2015-04-20 2017-11-02 Epizyme, Inc. Combination therapy for treating cancer
US20180311251A1 (en) 2015-06-10 2018-11-01 Epizyme, Inc. Ezh2 inhibitors for treating lymphoma
US10493076B2 (en) 2015-08-24 2019-12-03 Epizyme, Inc. Method for treating cancer
TW201718598A (zh) 2015-08-27 2017-06-01 美國禮來大藥廠 Ｅｚｈ２抑制劑
WO2017040190A1 (fr) 2015-08-28 2017-03-09 Constellation Pharmaceuticals, Inc. Formes cristallines de (r)-n-((4-méthoxy-6-méthyl-2-oxo-1,2-dihydropyridin-3-yl)méthyl)-2-méthyl-1-(1-(1-(2,2,2-trifluoroéthyl)pipéridin-4-yl)éthyl)-1h-indole-3-carboxamide
JP2019503391A (ja) 2016-01-29 2019-02-07 エピザイム，インコーポレイティド 癌を処置するための併用療法
AU2017273726B2 (en) * 2016-06-01 2023-10-19 Epizyme, Inc. Use of EZH2 inhibitors for treating cancer
MA45406A (fr) 2016-06-17 2019-04-24 Epizyme Inc Inhibiteurs d'ezh2 pour traiter le cancer
US10457640B2 (en) 2016-10-19 2019-10-29 Constellation Pharmaceuticals, Inc. Synthesis of inhibitors of EZH2
US11214561B2 (en) * 2017-01-25 2022-01-04 Ancureall Pharmaceutical (Shanghai) Co., Ltd. Histone methyltransferase EZH2 inhibitor, preparation method and pharmaceutical use thereof
WO2018183885A1 (fr) 2017-03-31 2018-10-04 Epizyme, Inc. Multithérapie pour le traitement du cancer
CN110944628A (zh) 2017-06-02 2020-03-31 Epizyme股份有限公司 使用ezh2抑制剂治疗癌症
CA3074720A1 (fr) 2017-09-05 2019-03-14 Epizyme, Inc. Polytherapie pour le traitement du cancer
EP3823671B1 (fr) 2018-07-09 2024-02-07 Fondation Asile Des Aveugles Inhibition de sous-unités de prc2 permettant de traiter des troubles oculaires
EP4017527A1 (fr) 2019-08-22 2022-06-29 Juno Therapeutics, Inc. Polythérapie basée sur une thérapie par lymphocytes t et un inhibiteur de protéine-2 homologue de l'activateur de zeste (ezh2) et procédés associés
IL294108A (en) * 2019-12-20 2022-08-01 Epizyme Inc Crystalline salt of hydrobromide of ezh2 inhibitor, its preparation and effective pharmaceutical composition for cancer treatment
WO2022208552A1 (fr) * 2021-03-31 2022-10-06 Msn Laboratories Private Limited, R&D Center Formes cristallines du [1,1'-biphényl]-3-carboxamide, n-[(1,2-dihydro-4,6-diméthyl-2-oxo-3-pyridinyl)méthyl]-5-[éthyl(tétrahydro-2h-pyran-4-yl)amino]-4-méthyl-4'-(4-morpholinylméthyl)-, bromhydrate (1:1) et son procédé de préparation

Family Cites Families (59)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0733729A (ja) 1993-07-26 1995-02-03 Kirin Brewery Co Ltd Ｎ−シアノ−ｎ′−置換−アリールカルボキシイミダミド化合物の製造法
AU694465B2 (en) 1993-12-27 1998-07-23 Eisai Co. Ltd. Anthranilic acid derivative
DE19516776A1 (de) 1995-05-10 1996-11-14 Boehringer Ingelheim Int Chromatin-Regulatorgene
US5741819A (en) 1995-06-07 1998-04-21 3-Dimensional Pharmaceuticals, Inc. Arylsulfonylaminobenzene derivatives and the use thereof as factor Xa inhibitors
JP3906935B2 (ja) 1995-12-18 2007-04-18 杏林製薬株式会社 Ｎ−置換ジオキソチアゾリジルベンズアミド誘導体及びその製造法
JP2002525352A (ja) 1998-09-30 2002-08-13 ザ、プロクター、エンド、ギャンブル、カンパニー ２−置換ケトアミド
UA71587C2 (uk) 1998-11-10 2004-12-15 Шерінг Акцієнгезелльшафт Аміди антранілової кислоти та їхнє застосування як лікарських засобів
US6710058B2 (en) 2000-11-06 2004-03-23 Bristol-Myers Squibb Pharma Company Monocyclic or bicyclic carbocycles and heterocycles as factor Xa inhibitors
KR100828982B1 (ko) 2000-12-28 2008-05-14 시오노기세이야쿠가부시키가이샤 칸나비노이드 2형 수용체 친화 작용을 갖는 피리돈 유도체
US7700293B2 (en) 2001-08-02 2010-04-20 The Regents Of The University Of Michigan Expression profile of prostate cancer
TW200303304A (en) * 2002-02-18 2003-09-01 Astrazeneca Ab Chemical compounds
WO2003070277A1 (fr) 2002-02-19 2003-08-28 Shionogi & Co., Ltd. Antiprurigineux
TW200306155A (en) 2002-03-19 2003-11-16 Du Pont Benzamides and advantageous compositions thereof for use as fungicides
US7442685B2 (en) 2003-06-13 2008-10-28 The University Of North Carolina At Chapel Hill DOT1 histone methyltransferases as a target for identifying therapeutic agents for leukemia
WO2005095386A1 (fr) 2004-03-30 2005-10-13 Chiron Corporation Derives du thiophene substitues en tant qu'agents anticancereux
WO2005118796A2 (fr) 2004-06-01 2005-12-15 The University Of North Carolina At Chapel Hill Complexe d'histone methyltransferase reconstitue et methodes associees d'identification de modulateurs
JO2787B1 (en) 2005-04-27 2014-03-15 امجين إنك, Alternative amide derivatives and methods of use
WO2006130720A2 (fr) 2005-06-02 2006-12-07 University Of North Carolina At Chapel Hill Purification, caracterisation et reconstitution d'une ubiquitine e3 ligase
FR2889526B1 (fr) 2005-08-04 2012-02-17 Aventis Pharma Sa 7-aza-indazoles substitues, compositions les contenant, procede de fabrication et utilisation
PT1940821E (pt) 2005-10-19 2013-05-29 Gruenenthal Gmbh Novos ligandos do receptor de vanilóide e seu uso para a produção de medicamentos
KR20080057296A (ko) 2005-10-21 2008-06-24 머크 앤드 캄파니 인코포레이티드 칼륨 채널 억제제
EP1941060B1 (fr) 2005-10-28 2012-02-29 The University of North Carolina At Chapel Hill Protéine déméthylase à domaine jmjc
EP1966141A1 (fr) 2005-12-14 2008-09-10 Brystol-Myers Squibb Company Heterocycles a six elements utiles en tant qu inhibiteurs de serine protease
AU2006336552B2 (en) 2006-01-20 2012-12-20 The University Of North Carolina At Chapel Hill Diagnostic and therapeutic targets for leukemia
MX2008014616A (es) 2006-05-15 2008-11-28 Irm Llc Compuestos y composiciones de tereftalamato, y su uso como inhibidores de integrasa de vih.
CA2652634A1 (fr) 2006-05-18 2007-11-29 Amphora Discovery Corporation Certaines quinolones substituees, compositions et utilisations
US8022246B2 (en) 2006-10-10 2011-09-20 The Burnham Institute For Medical Research Neuroprotective compositions and methods
JP2010519204A (ja) 2007-02-16 2010-06-03 アムジエン・インコーポレーテツド 窒素含有複素環ケトン類およびそれらのｃ−Ｍｅｔ阻害薬としての使用
EP2137158A4 (fr) 2007-02-28 2012-04-18 Methylgene Inc Inhibiteurs à petite molécule de protéines arginine méthyltransférases (prmt)
WO2008113006A1 (fr) 2007-03-14 2008-09-18 Xenon Pharmaceuticals Inc. Procédés d'utilisation de composés à base de quinolinone dans le traitement des maladies ou des affections associées aux canaux sodiques
DE102007017884A1 (de) 2007-04-13 2008-10-16 Grünethal GmbH Neue Vanilloid-Rezeptor Liganden und ihre Verwendung zur Herstellung von Arzneimitteln
WO2009006577A2 (fr) 2007-07-03 2009-01-08 The Regents Of The University Of Michigan Compositions et méthodes pour inhiber la protéine ezh2
DE102007047737A1 (de) * 2007-10-05 2009-04-30 Merck Patent Gmbh Piperidin- und Piperazinderivate
WO2009058298A1 (fr) 2007-10-31 2009-05-07 Merck & Co., Inc. Antagonistes du récepteur p2x3 utilisés dans le traitement de la douleur
BRPI0821227A2 (pt) 2007-12-19 2015-06-16 Cancer Rec Tech Ltd Composto, composição farmacêutica, método para preparar a mesma, uso de um composto, método para tratar uma doença ou distúrbio, para inibir função de raf e para inibir proliferação celular, inibir progressão do ciclo celular, promover apoptose, ou uma combinação de um ou mais dos mesmos
WO2009124137A2 (fr) 2008-04-01 2009-10-08 Mount Sinai School Of Medicine Of New York University Procédé de suppression d’une transcription de gène par méthylation d’histone lysine
US20100113415A1 (en) 2008-05-29 2010-05-06 Rajapakse Hemaka A Epha4 rtk inhibitors for treatment of neurological and neurodegenerative disorders and cancer
US8299093B2 (en) 2008-08-08 2012-10-30 New York Blood Center, Inc. Small molecule inhibitors of retroviral assembly and maturation
FR2934995B1 (fr) 2008-08-14 2010-08-27 Sanofi Aventis Composes d'azetidines polysubstitues, leur preparation et leur application en therapeutique
EP2411007A4 (fr) 2009-03-27 2012-12-05 Uab Research Foundation Modulation de la traduction à médiation par les séquences ires
US20110021362A1 (en) 2009-07-20 2011-01-27 Constellation Pharmaceuticals Agents for stimulating activity of methyl modifying enzymes and methods of use thereof
EP2521558A4 (fr) 2009-12-30 2013-07-03 Avon Prod Inc Composition éclaircissante topique et utilisations de celle-ci
WO2011140325A1 (fr) * 2010-05-07 2011-11-10 Glaxosmithkline Llc Indazoles
SI2566327T1 (sl) * 2010-05-07 2017-07-31 Glaxosmithkline Llc Indoli
US8637509B2 (en) 2010-05-07 2014-01-28 Glaxosmithkline Llc Azaindazoles
JP6389036B2 (ja) 2010-09-10 2018-09-12 エピザイム インコーポレイテッド ヒトｅｚｈ２の阻害剤、およびその使用方法
US9175331B2 (en) 2010-09-10 2015-11-03 Epizyme, Inc. Inhibitors of human EZH2, and methods of use thereof
WO2012068589A2 (fr) 2010-11-19 2012-05-24 Constellation Pharmaceuticals Modulateurs d'enzymes de modification par méthylation, compositions et utilisations associées
US8765792B2 (en) 2010-12-01 2014-07-01 Glaxosmithkline Llc Indoles
EP2646454B1 (fr) 2010-12-03 2015-07-08 Epizyme, Inc. Modulateurs à base de 7-déazapurine de l'histone méthyltransférase et procédés d'utilisation de ceux-ci
JP2014511389A (ja) 2011-02-28 2014-05-15 エピザイム インコーポレイテッド 置換６，５−縮合二環式ヘテロアリール化合物
TWI598336B (zh) 2011-04-13 2017-09-11 雅酶股份有限公司 經取代之苯化合物
JO3438B1 (ar) 2011-04-13 2019-10-20 Epizyme Inc مركبات بنزين مستبدلة بأريل أو أريل غير متجانس
AU2013232229B2 (en) 2012-03-12 2017-11-23 Epizyme, Inc. Inhibitors of human EZH2, and methods of use thereof
HUE060881T2 (hu) * 2012-04-13 2023-04-28 Epizyme Inc Sóforma az EZH2 gátláshoz
RU2019126908A (ru) 2012-04-13 2020-01-13 Эпизайм, Инк. Комбинированная терапия для лечения рака
US9562041B2 (en) 2012-05-16 2017-02-07 Glaxosmithkline Llc Enhancer of zeste homolog 2 inhibitors
CA2887243C (fr) 2012-10-15 2024-04-09 Epizyme, Inc. Methodes de traitement du cancer
PL3057962T3 (pl) 2013-10-16 2024-01-29 Epizyme, Inc. Postać soli chlorowodorku do inhibicji ezh2

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None *

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MX2014012380A (es) 2015-07-23
PL2836491T3 (pl) 2017-08-31
CN104603130B (zh) 2018-04-27
AU2018200168B2 (en) 2019-08-29
JP6255382B2 (ja) 2017-12-27
US20230140327A1 (en) 2023-05-04
KR102438340B1 (ko) 2022-08-30
EP2836491A1 (fr) 2015-02-18
IL266165A (en) 2019-06-30
CN104603130A (zh) 2015-05-06
RU2014145544A (ru) 2016-06-10
IL282732B2 (en) 2023-04-01
JP6634058B2 (ja) 2020-01-22
IL282732A (en) 2021-06-30

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